scholarly journals Molecular Cloning and Functional Expression inLactobacillus plantarum 80 of xylT, Encoding thed-Xylose–H+ Symporter ofLactobacillus brevis

1998 ◽  
Vol 64 (12) ◽  
pp. 4720-4728 ◽  
Author(s):  
Stéphane Chaillou ◽  
Yeou-Cherng Bor ◽  
Carl A. Batt ◽  
Pieter W. Postma ◽  
Peter H. Pouwels
Keyword(s):  
Xylose Isomerase ◽  
Proton Motive Force ◽  
Motive Force ◽  
Affinity Constant ◽  
Transport Activity ◽  
Gene Encoding ◽  
Kinase Gene ◽  
E Coli ◽  
Xylose Transport ◽  
Xylulose Kinase

ABSTRACT A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (encoding d-xylose isomerase), was cloned in Escherichia coli TG1. The sequence revealed two open reading frames which could code for the d-xylulose kinase gene (xylB) and another gene (xylT) encoding a protein of 457 amino acids with significant similarity to thed-xylose–H+ symporters of E. coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the d-xylose–Na+ symporter ofTetragenococcus halophila, XylE (57%), and to thel-arabinose–H+ symporter of E. coli, AraE (60%). The L. brevis xylABT genes showed an arrangement similar to that of the B. megaterium xylABT operon and the T. halophila xylABE operon. Southern hybridization performed with the Lactobacillus pentosus xylR gene (encoding the d-xylose repressor protein) as a probe revealed the existence of a xylR homologue inL. brevis which is not located with thexyABT locus. The existence of a functional XylR was further suggested by the presence of xylO sequences upstream ofxylA and xylT and by the requirement ofd-xylose for the induction of d-xylose isomerase, d-xylulose kinase, and d-xylose transport activities in L. brevis. When L. brevis was cultivated in a mixture of d-glucose andd-xylose, the d-xylose isomerase andd-xylulose kinase activities were reduced fourfold and thed-xylose transport activity was reduced by sixfold, suggesting catabolite repression by d-glucose ofd-xylose assimilation. The xylT gene was functionally expressed in Lactobacillus plantarum 80, a strain which lacks proton motive force-linked d-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of d-xylose in L. plantarum80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of d-glucose. The apparent affinity constant of XylT ford-xylose was approximately 215 μM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-d-glucose inhibited the transport ofd-xylose by XylT competitively, with aKi of 220 μM.

scholarly journals Blue light is a universal tactic signal forEscherichia coli

2017 ◽  
Author(s):  
Tatyana Perlova ◽  
Martin Gruebele ◽  
Yann R. Chemla
Keyword(s):  
Blue Light ◽  
Light Exposure ◽  
Biological Significance ◽  
Proton Motive Force ◽  
Motive Force ◽  
Swimming Velocity ◽  
Bacterial Strains ◽  
E Coli ◽  
Before And After ◽  
Swimming Bacteria

AbstractBlue light has been shown to elicit a tumbling response inE. coli, a non-phototrophic bacterium. The exact mechanism of this phototactic response is still unknown, and its biological significance remains unclear. Here, we quantify phototaxis inE. coliby analyzing single-cell trajectories in populations of free-swimming bacteria before and after light exposure. Bacterial strains expressing only one type of chemoreceptor reveal that all fiveE. colireceptors - Aer, Tar, Tsr, Tap and Trg - are capable of mediating a response to light. In particular, light exposure elicits a running response in Tap-only strain, the opposite of the tumbling response observed for all other strains. Light therefore emerges as a universal stimulus for allE. colichemoreceptors. We also show that blue light exposure causes a reversible decrease in swimming velocity, a proxy for proton motive force. We hypothesize that rather than sensing light directly, chemoreceptors sense light-induced perturbations in proton motive force.ImportanceOur findings provide new insights on the mechanism ofE. coliphototaxis, showing that all five chemoreceptor types respond to light and that their interactions play an important role in cell behavior. Our results also open up new avenues for examining and manipulatingE. colitaxis. Since light is a universal stimulus, it may provide a way to quantify interactions between different types of receptors. Since light is easier to control spatially and temporally than chemicals, it may be used to study swimming behavior in complex environments. Since phototaxis can cause migration ofE. colibacteria in light gradients, light may be used to control bacterial density for studying density-dependent processes in bacteria.

scholarly journals Mechanism of Bactericidal Activity of Microcin L inEscherichia coliandSalmonella enterica

2010 ◽  
Vol 55 (3) ◽  
pp. 997-1007 ◽  
Author(s):  
Natacha Morin ◽  
Isabelle Lanneluc ◽  
Nathalie Connil ◽  
Marie Cottenceau ◽  
Anne Marie Pons ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Bactericidal Activity ◽  
Salmonella Enterica ◽  
Cytoplasmic Membrane ◽  
Genetic System ◽  
Proton Motive Force ◽  
Motive Force ◽  
Membrane Properties ◽  
Outer Membrane Receptor ◽  
E Coli

ABSTRACTFor the first time, the mechanism of action of microcin L (MccL) was investigated in live bacteria. MccL is a gene-encoded peptide produced byEscherichia coliLR05 that exhibits a strong antibacterial activity against relatedEnterobacteriaceae, includingSalmonella entericaserovars Typhimurium and Enteritidis. We first subcloned the MccL genetic system to remove the sequences not involved in MccL production. We then optimized the MccL purification procedure to obtain large amounts of purified microcin to investigate its antimicrobial and membrane properties. We showed that MccL did not induce outer membrane permeabilization, which indicated that MccL did not use this way to kill the sensitive cell or to enter into it. Using a set ofE. coliandSalmonella entericamutants lacking iron-siderophore receptors, we demonstrated that the MccL uptake required the outer membrane receptor Cir. Moreover, the MccL bactericidal activity was shown to depend on the TonB protein that transduces the proton-motive force of the cytoplasmic membrane to transport iron-siderophore complexes across the outer membrane. Using carbonyl cyanide 3-chlorophenylhydrazone, which is known to fully dissipate the proton-motive force, we proved that the proton-motive force was required for the bactericidal activity of MccL onE. coli. In addition, we showed that a primary target of MccL could be the cytoplasmic membrane: a high level of MccL disrupted the inner membrane potential ofE. colicells. However, no permeabilization of the membrane was detected.

scholarly journals Blue Light Is a Universal Signal forEscherichia coliChemoreceptors

2019 ◽  
Vol 201 (11) ◽  
Author(s):  
Tatyana Perlova ◽  
Martin Gruebele ◽  
Yann R. Chemla
Keyword(s):  
Blue Light ◽  
Light Exposure ◽  
Proton Motive Force ◽  
Motive Force ◽  
Swimming Velocity ◽  
Bacterial Strains ◽  
Content Type ◽  
E Coli ◽  
Before And After ◽  
Swimming Bacteria

ABSTRACTBlue light has been shown to elicit a tumbling response inEscherichia coli, a nonphototrophic bacterium. The exact mechanism of this phototactic response is still unknown. Here, we quantify phototaxis inE. coliby analyzing single-cell trajectories in populations of free-swimming bacteria before and after light exposure. Bacterial strains expressing only one type of chemoreceptor reveal that all fiveE. colireceptors (Aer, Tar, Tsr, Tap, and Trg) are capable of mediating responses to light. In particular, light exposure elicits a running response in the Tap-only strain, the opposite of the tumbling responses observed for all other strains. Therefore, light emerges as a universal stimulus for allE. colichemoreceptors. We also show that blue light exposure causes a reversible decrease in swimming velocity, a proxy for proton motive force. This result is consistent with a previously proposed hypothesis that, rather than sensing light directly, chemoreceptors sense light-induced perturbations in proton motive force, although other factors are also likely to contribute.IMPORTANCEOur findings provide new insights into the mechanism ofE. coliphototaxis, showing that all five chemoreceptor types respond to light and their interactions play an important role in cell behavior. Our results also open up new avenues for examining and manipulatingE. colitaxis. Since light is a universal stimulus, it may provide a way to quantify interactions among different types of receptors. Because light is easier to control spatially and temporally than chemicals, it may be used to study swimming behavior in complex environments. Since phototaxis can cause migration ofE. colibacteria in light gradients, light may be used to control bacterial density for studying density-dependent processes in bacteria.

scholarly journals Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

2006 ◽  
Vol 72 (5) ◽  
pp. 3418-3428 ◽  
Author(s):  
Hideo Kawaguchi ◽  
Alain A. Vert�s ◽  
Shohei Okino ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Xylose Isomerase ◽  
Mineral Medium ◽  
Gene Encoding ◽  
Xylose Consumption ◽  
E Coli ◽  
High Copy Number Plasmid ◽  
Sugar Mixture ◽  
Pentose Sugar

ABSTRACT The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vert�s, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.

Characterization and Functional Expression of Xylose Isomerase from Thermus thermophilus

2013 ◽  
Vol 641-642 ◽  
pp. 919-922
Author(s):  
An Gen Lu ◽  
Ze Xi Yang ◽  
Fei Wang ◽  
Lang Xu ◽  
Wen Ying Deng ◽  
...  
Keyword(s):  
Fossil Fuels ◽  
Specific Activity ◽  
Thermus Thermophilus ◽  
Xylose Isomerase ◽  
Functional Expression ◽  
Future Application ◽  
Gene Encoding ◽  
E Coli ◽  
Rate Limiting ◽  
Pentose Sugars

Ethanol produced from hexose and pentose sugars hydrolysated by lignocellulose is an environment-friendly alternative to fossil fuels. Xylose isomerase is the major rate-limiting enzyme in the ethanol synthesis biologically pathway of xylose fermentation. In present study, xylA gene encoding xylose isomerase was cloned from Thermus thermophilus and overexpressed in E. coli BL21. Purified recombinant enzyme was used to study the enzymatic characterization. Specific activity of recombinant PDOR was 19.6 U/mg. Optimal temperature and pH were 80 °C, 8.0, respectively. Km and Vmax values were 15.9 mM, 22.8 U/mg. This research may form a basis for the future application of xylose isomerase.

Probing the dynamics of the proton-motive force in E. coli

2014 ◽  
Author(s):  
Tom J. Zajdel ◽  
Michaela A. TerAvest ◽  
Behzad Rad ◽  
Caroline M. Ajo-Franklin ◽  
Michel M. Maharbiz
Keyword(s):  
Proton Motive Force ◽  
Motive Force ◽  
E Coli

scholarly journals Membrane Transporters of the Major Facilitator Superfamily Are Essential for Long-Term Maintenance of Phenotypic Tolerance to Multiple Antibiotics in E. coli

2021 ◽  
Author(s):  
Yingkun Wan ◽  
Miaomiao Wang ◽  
Edward Wai Chi Chan ◽  
Sheng Chen
Keyword(s):  
Proton Motive Force ◽  
Membrane Transporters ◽  
Motive Force ◽  
Major Facilitator Superfamily ◽  
Starvation Stress ◽  
E Coli ◽  
Major Facilitator ◽  
Term Maintenance ◽  
Multiple Antibiotics

We recently showed that the antibiotic-tolerant subpopulation of bacteria or persisters actively maintain the transmembrane proton motive force (PMF) to survive starvation stress for a prolonged period. This work further shows that the reason why antibiotic persisters need to maintain PMF is that PMF is required to support a range of efflux or transportation functions.

scholarly journals Genetic Evidence for a Defective Xylan Degradation Pathway in Lactococcus lactis

2001 ◽  
Vol 67 (4) ◽  
pp. 1445-1452 ◽  
Author(s):  
Karn A. Erlandson ◽  
Soazig C. Delamarre ◽  
Carl A. Batt
Keyword(s):  
Lactococcus Lactis ◽  
Xylose Isomerase ◽  
Degradation Pathway ◽  
Open Reading Frames ◽  
Northern Hybridization ◽  
Xylose Metabolism ◽  
Kinase Gene ◽  
Reading Frame ◽  
Xylan Degradation ◽  
Xylulose Kinase

ABSTRACT Genetic and biochemical evidence for a defective xylan degradation pathway was found linked to the xylose operon in three lactococcal strains, Lactococcus lactis 210, L. lactis IO-1, and L. lactis NRRL B-4449. Immediately downstream of the xylulose kinase gene (xylB) (K. A. Erlandson, J.-H. Park, W. El Khal, H.-H. Kao, P. Basaran, S. Brydges, and C. A. Batt, Appl. Environ. Microbiol. 66:3974–3980, 1999) are two open reading frames encoding a mutarotase (xylM) and a xyloside transporter (xynT) and a partial open reading frame encoding a β-xylosidase (xynB). These are functions previously unreported for lactococci or lactobacilli. The mutarotase activity of the putative xylM gene product was confirmed by overexpression of the L. lactis enzyme in Escherichia coli and purification of recombinant XylM. We hypothesize that the mutarotase links xylan degradation to xylose metabolism due to the anomeric preference of xylose isomerase. In addition, Northern hybridization experiments suggested that the xylM and xynTB genes are cotranscribed with the xylRAB genes, responsible for xylose metabolism. Although none of the three strains appeared to metabolize xylan or xylobiose, they exhibited xylosidase activity, and L. lactis IO-1 and L. lactis NRRL B-4449 had functional mutarotases.

scholarly journals The gusBC Genes of Escherichia coli Encode a Glucuronide Transport System

2005 ◽  
Vol 187 (7) ◽  
pp. 2377-2385 ◽  
Author(s):  
Wei-Jun Liang ◽  
Kate J. Wilson ◽  
Hao Xie ◽  
Jan Knol ◽  
Shun'ichi Suzuki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Expression Vector ◽  
Proton Motive Force ◽  
Mammalian Host ◽  
Transport Activity ◽  
Secondary Active Transport ◽  
E Coli ◽  
Fecal Isolate ◽  
Vector Transport

ABSTRACT Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of β-glucuronides with synthetic [14C]phenyl-1-thio-β-d-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the E. coli genome, and their expression is induced by a variety of β-d-glucuronides. Measurements of transport in right-side-out subcellular vesicles show the system has the characteristics of secondary active transport energized by the respiration-generated proton motive force. When the genes were cloned together downstream of the tac operator-promoter in the plasmid pTTQ18 expression vector, transport activity was increased considerably with isopropylthiogalactopyranoside as the inducer. Amplified expression of the GusB and GusC proteins enabled visualization and identification by N-terminal sequencing of both proteins, which migrated at ca. 32 kDa and 44 kDa, respectively. Separate expression of the GusB protein showed that it is essential for glucuronide transport and is located in the inner membrane, while the GusC protein does not catalyze transport but assists in an as yet unknown manner and is located in the outer membrane. The output of glucuronides as waste by mammals and uptake for nutrition by gut bacteria or reabsorption by the mammalian host is discussed.

Sign in / Sign up

Export Citation Format

Share Document