Substrate specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa

1993 ◽  
Vol 39 (7) ◽  
pp. 722-725 ◽  
Author(s):  
John L. Wylie ◽  
Elizabeth A. Worobec
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Glucose Transport ◽  
Transport System ◽  
Binding Protein ◽  
High Affinity ◽  
Glucose Binding ◽  
Glucose Transport System ◽  
Glucose Binding Protein

Specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa was examined. At a concentration of [14C]glucose near the Vmax of the system, inhibition by maltose, galactose, and xylose was detected. This inhibition is similar to that detected in earlier in vivo studies and correlates with the known specificity of OprB, a glucose-specific porin of P. aeruginosa. At a level of [14C]glucose 100 times lower, only unlabelled glucose inhibited uptake to any extent. This matches the known in vitro specificity of the periplasmic glucose binding protein. These findings were used to explain the discrepancy between earlier in vivo and in vitro results reported in the literature.Key words: Pseudomonas aeruginosa, glucose transport, OprB, glucose binding protein.

Reconstitution of glucose uptake and chemotaxis in Pseudomonas aeruginosa glucose transport defective mutants

1993 ◽  
Vol 39 (11) ◽  
pp. 1079-1083 ◽  
Author(s):  
Laura M. Sly ◽  
Elizabeth A. Worobec ◽  
Richard E. Perkins ◽  
Paul V. Phibbs Jr.
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Binding Protein ◽  
Cell Uptake ◽  
Wild Type ◽  
Binding Assays ◽  
Glucose Binding ◽  
Glucose Transport System ◽  
Glucose Binding Protein

Wild-type glucose uptake and glucose chemotaxis activities were restored in glucose transport defective Pseudomonas aeruginosa strains PFB360 and PFB362 after introduction of plasmid pPZ129, containing a 1.1-kilobase DNA fragment that is essential for the expression of the P. aeruginosa periplasmic glucose binding protein. The restoration of glucose uptake and chemotaxis to wild-type levels in these strains was also achieved by reconstitution with cold-shock fluid and purified glucose binding protein isolated from P. aeruginosa PA01 wild-type strain H103 grown in conditions resulting in the induction of the high-affinity glucose transport system. Glucose uptake was determined by whole cell uptake and shock fluid binding of D-[U-14C]glucose, using standard filter binding assays. Positive chemotaxis towards glucose was assessed by capillary assays using 10 mM glucose, the amount required for optimal chemotaxis, and judged by plating capillary contents accumulated after 30 min.Key words: glucose-binding protein, Pseudomonas aeruginosa, glucose transport, chemotaxis.

Properties and formation of the squash high-affinity glucose transport system

1970 ◽  
Vol 48 (9) ◽  
pp. 1515-1520 ◽  
Author(s):  
Joseph G. Hancock
Keyword(s):  
Glucose Transport ◽  
Transport System ◽  
Concentration Gradient ◽  
Indoleacetic Acid ◽  
Cucurbita Maxima ◽  
Michaelis Constant ◽  
High Affinity ◽  
Glucose Transport System

Uptake of 3-o-methylglucose (MeG) by excised squash (Cucurbita maxima Dcne) hypocotyl sections and other plants increased greatly with age. A high-affinity (HA) transport system, with an apparent Michaelis constant (Km) of 0.6–0.8 mM, was formed during ageing in squash sections and was probably responsible for the enhanced MeG uptake. Net transport occurred against a concentration gradient in aged tissues. Accumulation of MeG by freshly excised sections, which possessed a low-affinity (LA) transport system (Km, 25–45 mM), was negligible. Glucose competitively inhibited MeG uptake by both the LA and HA systems and appeared to be the preferred substrate in vivo. When sections were bathed in glucose or galactose, an efflux of preabsorbed MeG occurred. Though inhibited by UO22+, uptake of MeG by the HA system was not affected by Ca2+, Mg2+, or K+.Glucose (10 mM) and indoleacetic acid (> 0.1 mM) repressed the formation of the HA system, while kinetin, gibberellin, and indoleacetic acid at lower concentrations (< 0.01 mM) had no effect.

scholarly journals RCO-3 and COL-26 form an external-to-internal module that regulates the dual-affinity glucose transport system in Neurospora crassa

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Jinyang Li ◽  
Qian Liu ◽  
Jingen Li ◽  
Liangcai Lin ◽  
Xiaolin Li ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Neurospora Crassa ◽  
Glucose Transport ◽  
Transport System ◽  
Rational Design ◽  
Glucose Sensor ◽  
Glucose Transporters ◽  
Glucose Fluctuation ◽  
High Affinity ◽  
Glucose Transport System

Abstract Background Low- and high-affinity glucose transport system is a conserved strategy of microorganism to cope with environmental glucose fluctuation for their growth and competitiveness. In Neurospora crassa, the dual-affinity glucose transport system consists of a low-affinity glucose transporter GLT-1 and two high-affinity glucose transporters HGT-1/HGT-2, which play diverse roles in glucose transport, carbon metabolism, and cellulase expression regulation. However, the regulation of this dual-transporter system in response to environmental glucose fluctuation is not yet clear. Results In this study, we report that a regulation module consisting of a downstream transcription factor COL-26 and an upstream non-transporting glucose sensor RCO-3 regulates the dual-affinity glucose transport system in N. crassa. COL-26 directly binds to the promoter regions of glt-1, hgt-1, and hgt-2, whereas RCO-3 is an upstream factor of the module whose deletion mutant resembles the Δcol-26 mutant phenotypically. Transcriptional profiling analysis revealed that Δcol-26 and Δrco-3 mutants had similar transcriptional profiles, and both mutants had impaired response to a glucose gradient. We also showed that the AMP-activated protein kinase (AMPK) complex is involved in regulation of the glucose transporters. AMPK is required for repression of glt-1 expression in starvation conditions by inhibiting the activity of RCO-3. Conclusions RCO-3 and COL-26 form an external-to-internal module that regulates the glucose dual-affinity transport system. Transcription factor COL-26 was identified as the key regulator. AMPK was also involved in the regulation of the dual-transporter system. Our findings provide novel insight into the molecular basis of glucose uptake and signaling in filamentous fungi, which may aid in the rational design of fungal strains for industrial purposes.

scholarly journals Comparison of intestinal glucose flux and electrogenic current demonstrates two absorptive pathways in pig and one in Nile tilapia and rainbow trout

2020 ◽  
Vol 318 (2) ◽  
pp. R245-R255
Author(s):  
Marina Subramaniam ◽  
Cole B. Enns ◽  
Khanh Luu ◽  
Lynn P. Weber ◽  
Matthew E. Loewen
Keyword(s):  
Rainbow Trout ◽  
Glucose Transport ◽  
Transport System ◽  
Fish Species ◽  
Nile Tilapia ◽  
High Capacity ◽  
Electrogenic Transport ◽  
High Affinity ◽  
Glucose Transport System ◽  
Total Glucose

The mucosal-to-serosal flux of 14C 3- O-methyl-d-glucose was compared against the electrogenic transport of d-glucose across ex vivo intestinal segments of Nile tilapia, rainbow trout, and pig in Ussing chambers. The difference in affinities ( Km “fingerprints”) between pig flux and electrogenic transport of glucose, and the absence of this difference in tilapia and trout, suggest two absorptive pathways in the pig and one in the fish species examined. More specifically, the total mucosal-to-serosal flux revealed a super high-affinity, high-capacity (sHa/Hc) total glucose transport system in tilapia; a super high-affinity, low-capacity (sHa/Lc) total glucose transport system in trout and a low-affinity, low-capacity (La/Lc) total glucose transport system in pig. Comparatively, electrogenic glucose absorption revealed similar Km in both fish species, with a super high-affinity, high capacity (sHa/Hc) system in tilapia; a super high-affinity/super low-capacity (sHa/sLc) system in trout; but a different Km fingerprint in the pig, with a high-affinity, low-capacity (Ha/Lc) system. This was supported by different responses to inhibitors of sodium-dependent glucose transporters (SGLTs) and glucose transporter type 2 (GLUT2) administered on the apical side between species. More specifically, tilapia flux was inhibited by SGLT inhibitors, but not the GLUT2 inhibitor, whereas trout lacked response to inhibitors. In contrast, the pig responded to inhibition by both SGLT and GLUT2 inhibitors with a higher expression of GLUT2. Altogether, it would appear that two pathways are working together in the pig, allowing it to have continued absorption at high glucose concentrations, whereas this is not present in both tilapia and trout.

scholarly journals A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability.

1987 ◽  
Vol 7 (9) ◽  
pp. 3268-3276 ◽  
Author(s):  
A B Sachs ◽  
R W Davis ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Association Constant ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
High Affinity ◽  
Terminal Domain ◽  
Necessary And Sufficient

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

scholarly journals The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo.

1997 ◽  
Vol 17 (6) ◽  
pp. 3194-3201 ◽  
Author(s):  
R J Buckanovich ◽  
R B Darnell
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Motor Dysfunction ◽  
High Affinity ◽  
Loop Region ◽  
Nuclear Rna ◽  
Rna Ligands

Nova-1, an autoantigen in paraneoplastic opsoclonus myoclonus ataxia (POMA), a disorder associated with breast cancer and motor dysfunction, is a neuron-specific nuclear RNA binding protein. We have identified in vivo Nova-1 RNA ligands by combining affinity-elution-based RNA selection with protein-RNA immunoprecipitation. Starting with a pool of approximately 10(15) random 52-mer RNAs, we identified long stem-loop RNA ligands that bind to Nova-1 with high affinity (Kd of approximately 2 nM). The loop region of these RNAs harbors a approximately 15-bp pyrimidine-rich element [UCAU(N)(0-2)]3 which is essential for Nova-1 binding. Mutagenesis studies defined the third KH domain of Nova-1 and the [UCAU(N)(0-2)]3 element as necessary for in vitro binding. Consensus [UCAU (N)(0-2)], elements were identified in two neuronal pre-mRNAs, one encoding the inhibitory glycine receptor alpha2 (GlyR alpha2) and a second encoding Nova-1 itself. Nova-1 protein binds these RNAs with high affinity and specificity in vitro, and this binding can be blocked by POMA antisera. Moreover, both Nova-1 and GlyR alpha2 pre-mRNAs specifically coimmunoprecipitated with Nova-1 protein from brain extracts. Thus, Nova-1 functions as a sequence-specific nuclear RNA binding protein in vivo; disruption of the specific interaction between Nova-1 and GlyR alpha2 pre-mRNA may underlie the motor dysfunction seen in POMA.

A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability

1987 ◽  
Vol 7 (9) ◽  
pp. 3268-3276 ◽  
Author(s):  
A B Sachs ◽  
R W Davis ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Association Constant ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
High Affinity ◽  
Terminal Domain ◽  
Necessary And Sufficient

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

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