scholarly journals Archaeal Binding Protein-Dependent ABC Transporter: Molecular and Biochemical Analysis of the Trehalose/Maltose Transport System of the Hyperthermophilic Archaeon Thermococcus litoralis

1998 ◽  
Vol 180 (3) ◽  
pp. 680-689 ◽  
Author(s):  
Reinhold Horlacher ◽  
Karina B. Xavier ◽  
Helena Santos ◽  
Jocelyne DiRuggiero ◽  
Marina Kossmann ◽  
...  
Keyword(s):  
Transport System ◽  
Signal Sequence ◽  
Binding Protein ◽  
Inducible Promoter ◽  
Transport Systems ◽  
Thermococcus Litoralis ◽  
Hyperthermophilic Archaeon ◽  
E Coli ◽  
Transmembrane Segments ◽  
Maltose Transport

ABSTRACT We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeonThermococcus litoralis that is homologous to themalEFG cluster encoding the Escherichia colimaltose transport system. The deduced amino acid sequence of themalE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85°C with aKd of 0.16 μM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.

scholarly journals Biochemical evidence for the presence of two α-glucoside ABC-transport systems in the hyperthermophilic archaeonPyrococcus furiosus

Archaea ◽  
2002 ◽  
Vol 1 (1) ◽  
pp. 19-25 ◽  
Author(s):  
Sonja M. Koning ◽  
Wil N. Konings ◽  
Arnold J.M. Driessen
Keyword(s):  
Transport System ◽  
Pyrococcus Furiosus ◽  
Binding Protein ◽  
Transport Systems ◽  
Atp Binding ◽  
Hyperthermophilic Archaeon ◽  
Biochemical Evidence ◽  
Abc Transport ◽  
Maltose Transporter ◽  
Atp Binding Cassette

The hyperthermophilic archaeonPyrococcus furiosuscan utilize different carbohydrates, such as starch, maltose and trehalose. Uptake of α-glucosides is mediated by two different, binding protein-dependent, ATP-binding cassette (ABC)-type transport systems. The maltose transporter also transports trehalose, whereas the maltodextrin transport system mediates the uptake of maltotriose and higher malto-oligosaccharides, but not maltose. Both transport systems are induced during growth on their respective substrates.

scholarly journals High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis.

1996 ◽  
Vol 178 (16) ◽  
pp. 4773-4777 ◽  
Author(s):  
K B Xavier ◽  
L O Martins ◽  
R Peist ◽  
M Kossmann ◽  
W Boos ◽  
...  
Keyword(s):  
Transport System ◽  
Thermococcus Litoralis ◽  
Hyperthermophilic Archaeon ◽  
High Affinity

scholarly journals Overproduction of Penicillin-Binding Protein 2 and Its Inactive Variants Causes Morphological Changes and Lysis in Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 4975-4983 ◽  
Author(s):  
Blaine A. Legaree ◽  
Calvin B. Adams ◽  
Anthony J. Clarke
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Morphological Changes ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Prolonged Incubation ◽  
Lytic Transglycosylases ◽  
E Coli ◽  
Derivatives Of

ABSTRACT Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their N-terminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.

Periplasmic binding protein-dependent transport systems: the membrane-associated components

1990 ◽  
Vol 326 (1236) ◽  
pp. 353-365 ◽  
Keyword(s):  
Transport System ◽  
Atp Hydrolysis ◽  
Binding Protein ◽  
Primary Source ◽  
Energy Coupling ◽  
Transport Systems ◽  
Common Mechanism ◽  
Atp Binding ◽  
Periplasmic Binding Protein ◽  
Dependent Transport

Periplasmic binding protein-dependent transport systems are multicomponent, consisting of several inner membrane-associated proteins and a periplasmic component. The membrane-associated components of different systems are related in organization and function suggesting that, despite different substrate specificities, each transport system functions by a common mechanism. Current understanding of these components is reviewed. The nature of energy coupling to periplasmic transport systems has long been debated. Recent data now demonstrate that ATP hydrolysis is the primary source of energy for transport. The ATP-binding transport components are the best characterized of a family of closely related ATP-binding proteins believed to couple ATP hydrolysis to a variety of different biological processes. Intriguingly, systems closely related to periplasmic binding protein-dependent transport systems have recently been identified in several Gram-positive organisms (which lack a periplasm) and in eukaryotic cells. This class of transport system appears to be widespread in nature, serving a variety of important and diverse functions.

scholarly journals 2-Deoxy-d-galactose, a substrate for the galactose-transport system of Escherichia coli

1977 ◽  
Vol 168 (1) ◽  
pp. 15-22 ◽  
Author(s):  
P J F Henderson ◽  
R A Giddens
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Transport Systems ◽  
Mutual Inhibition ◽  
E Coli ◽  
System 2 ◽  
Galactose Transport ◽  
Proton Uptake ◽  
Inhibition Studies ◽  
Lactose Transport

The following observations showed that 2-deoxy-D-galactose is a useful tool for the isolation and elucidation of the activity of one system for galactose uptake into Escherichia coli. 1. 2-Deoxygalactose, which is not a substrate for growth of E. coli, was transported into strains of the organism induced for galactose transport. 2. By using appropriate mutants it was shown that 2-deoxygalactose is a much better substrate for the galactose-transport system than for the methyl galactoside-transport system. This was confirmed by the results of mutual inhibition studies with substrates of each transport system. 3. The glucose-, arabinose- or lactose-transport systems did not effect significant transport of 2-deoxygalactose. 4. Like other substrates of the galactose-transport system, 2-deoxygalactose promoted effective proton uptake into de-energized suspensions of appropriate E. coli strains. 5. The S183 series of E. coli mutants were found to contain a constitutive galactose-transport system, if 2-deoxygalactose transport is used as one criterion for such activity.

scholarly journals Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture

2000 ◽  
Vol 182 (24) ◽  
pp. 7029-7034 ◽  
Author(s):  
Artem Khlebnikov ◽  
Øystein Risa ◽  
Tove Skaug ◽  
Trent A. Carrier ◽  
J. D. Keasling
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
High Capacity ◽  
Inducible Promoter ◽  
Transport Systems ◽  
Specific Expression ◽  
Homogeneous Population ◽  
E Coli ◽  
Transport Control ◽  
Inducible Gene

ABSTRACT The arabinose-inducible promoter PBAD is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, thearaE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-β-d-thiogalactopyranoside-induciblePtac and Ptaclac promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independentaraE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (PBAD ) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGHmutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.

scholarly journals Identification of the nik Gene Cluster of Brucella suis: Regulation and Contribution to Urease Activity

2001 ◽  
Vol 183 (2) ◽  
pp. 426-434 ◽  
Author(s):  
Véronique Jubier-Maurin ◽  
Agnès Rodrigue ◽  
Safia Ouahrani-Bettache ◽  
Marion Layssac ◽  
Marie-Andrée Mandrand-Berthelot ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Transport System ◽  
Metal Ion ◽  
Urease Activity ◽  
Regulatory Gene ◽  
Transport Systems ◽  
Hydrogenase Activity ◽  
E Coli ◽  
Insertional Inactivation ◽  
Brucella Suis

ABSTRACT Analysis of a Brucella suis 1330 gene fused to agfp reporter, and identified as being induced in J774 murine macrophage-like cells, allowed the isolation of a gene homologous to nikA, the first gene of the Escherichia coli operon encoding the specific transport system for nickel. DNA sequence analysis of the corresponding B. suis niklocus showed that it was highly similar to that of E. coliexcept for localization of the nikR regulatory gene, which lies upstream from the structural nikABCDE genes and in the opposite orientation. Protein sequence comparisons suggested that the deduced nikABCDE gene products belong to a periplasmic binding protein-dependent transport system. ThenikA promoter-gfp fusion was activated in vitro by low oxygen tension and metal ion deficiency and was repressed by NiCl2 excess. Insertional inactivation of nikAstrongly reduced the activity of the nickel metalloenzyme urease, which was restored by addition of a nickel excess. Moreover, thenikA mutant of B. suis was functionally complemented with the E. coli nik gene cluster, leading to the recovery of urease activity. Reciprocally, an E. colistrain harboring a deleted nik operon recovered hydrogenase activity by heterologous complementation with the B. suis nik locus. Taking into account these results, we propose that thenik locus of B. suis encodes a nickel transport system. The results further suggest that nickel could enter B. suis via other transport systems. Intracellular growth rates of the B. suis wild-type and nikA mutant strains in human monocytes were similar, indicating that nikA was not essential for this step of infection. We discuss a possible role of nickel transport in maintaining enzymatic activities which could be crucial for survival of the bacteria under the environmental conditions encountered within the host.

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