Analysis of xylose operon regulation by Mud (Apr, lac) fusion: trans effect of plasmid coded xylose operon

1985 ◽  
Vol 31 (10) ◽  
pp. 930-933 ◽  
Author(s):  
C. A. Batt ◽  
M. S. Bodis ◽  
S. K. Picataggio ◽  
M. C. Claps ◽  
S. Jamas ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Element ◽  
Xylose Isomerase ◽  
Galactosidase Activity ◽  
Trans Effect ◽  
Isomerase Activity ◽  
Operon Regulation ◽  
Xyla Gene

The regulation of the Escherichia coli xylose operon was investigated by Mud (Apr, lac) fusion. β-Galactosidase activity from the xyl::Mud (Apr, lac) fusion was induced by xylose but not by arabinose and repressed by glucose. The chromosomal fusion was used to analyze the trans effect of plasmid borne xyl genes. The xylA gene repressed the expression of the xyl::Mud (Apr, lac) but xylose isomerase activity was not required for this repression. The results suggest that the regulation of the xylose operon may not be exclusively by a separate regulatory element but that the xylA gene may control the expression of the operon.

Lag, adaptation and ageing in a micro-organism ( Bact. lactis aerogenes )

1961 ◽  
Vol 155 (959) ◽  
pp. 195-201 ◽  
Keyword(s):  
Carbon Source ◽  
Generation Time ◽  
Sole Carbon Source ◽  
The Other ◽  
Lag Phase ◽  
Glucose Medium ◽  
Galactosidase Activity ◽  
Aerobacter Aerogenes ◽  
Isomerase Activity

The lag preceding growth of Bact. lactis aerogenes (Aerobacter aerogenes) after a first transfer to a medium containing D-arabinose as sole carbon source increases with the age and decreases with the size of the inoculum. During the long lag phase the β -galactosidase activity declines steeply. In contrast with this (and with a control ageing in a glucose medium) the D-ribulose isomerase activity is maintained, although no detectable consumption of D-arabinose occurs. If the long lag of unadapted cells in D-arabinose is divided into parts by intermediate passages in glucose or lactose media, the sum of the partial lags is nearly constant and equal to that observed when there is no interruption. But the periodic passages in the other media increase the rate at which growth eventually occurs in the D-arabinose. It is concluded that during the lag a decay of the enzymes in general occurs concomitantly with the development of the specific mechanisms concerned in the utilization of the new substrate. The balance of these processes (together with varying loss or retention of diffusible metabolites) is largely responsible for the observed variations in lag and mean generation time.

Regulation of RcsA by the ClpYQ (HslUV) protease in Escherichia coli

Microbiology ◽  
2004 ◽  
Vol 150 (2) ◽  
pp. 437-446 ◽  
Author(s):  
Mei-Shiue Kuo ◽  
Kuei-Peng Chen ◽  
Whi Fin Wu
Keyword(s):  
Escherichia Coli ◽  
Lon Protease ◽  
Specific Substrate ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Reporter Construct ◽  
Fusion Construct ◽  
In The Wild ◽  
A Cell ◽  
Cell Division Inhibitor

Escherichia coli ClpYQ protease and Lon protease possess a redundant function for degradation of SulA, a cell division inhibitor. An experimental cue implied that the capsule synthesis activator RcsA, a known substrate of Lon, is probably a specific substrate for the ClpYQ protease. This paper shows that overexpression of ClpQ and ClpY suppresses the mucoid phenotype of a lon mutant. Since the cpsB (wcaB) gene, involved in capsule synthesis, is activated by RcsA, the reporter construct cpsB–lacZ was used to assay for β-galactosidase activity and thus follow RcsA stability. The expression of cpsB–lacZ was increased in double mutants of lon in combination with clpQ or/and clpY mutation(s) compared with the wild-type or lon single mutants. Overproduction of ClpYQ or ClpQ decreased cpsB–lacZ expression. Additionally, a PBAD–rcsA fusion construct showed quantitatively that an inducible RcsA activates cpsB–lacZ expression. The effect of RcsA on cpsB–lacZ expression was shown to be influenced by the ClpYQ activities. Moreover, a rcsA Red –lacZ translational fusion construct showed higher activity of RcsARed–LacZ in a clpQ clpY strain than in the wild-type. By contrast, overproduction of cellular ClpYQ resulted in decreased β-galactosidase levels of RcsARed–LacZ. Taken together, the data indicate that ClpYQ acts as a secondary protease in degrading the Lon substrate RcsA.

Thermotoga neapolitana Homotetrameric Xylose Isomerase Is Expressed as a Catalytically Active and Thermostable Dimer in Escherichia coli

1998 ◽  
Vol 64 (7) ◽  
pp. 2357-2360 ◽  
Author(s):  
J. Michael Hess ◽  
Vladimir Tchernajenko ◽  
Claire Vieille ◽  
J. Gregory Zeikus ◽  
Robert M. Kelly
Keyword(s):  
Escherichia Coli ◽  
Differential Scanning Calorimetry ◽  
Gel Filtration ◽  
Xylose Isomerase ◽  
Thermotoga Neapolitana ◽  
Thermal Transitions ◽  
Scanning Calorimetry ◽  
Catalytically Active ◽  
The Stability ◽  
Tetrameric Form

ABSTRACT The xylA gene from Thermotoga neapolitana5068 was expressed in Escherichia coli. Gel filtration chromatography showed that the recombinant enzyme was both a homodimer and a homotetramer, with the dimer being the more abundant form. The purified native enzyme, however, has been shown to be exclusively tetrameric. The two enzyme forms had comparable stabilities when they were thermoinactivated at 95°C. Differential scanning calorimetry revealed thermal transitions at 99 and 109.5°C for both forms, with an additional shoulder at 91°C for the tetramer. These results suggest that the association of the subunits into the tetrameric form may have little impact on the stability and biocatalytic properties of the enzyme.

High sensitivity detection of Escherichia coli based on the measurement of β-galactosidase activity by microchip capillary electrophoresis combined with field-amplified sample injection

2019 ◽  
Vol 11 (11) ◽  
pp. 1558-1565 ◽  
Author(s):  
Yan Zhang ◽  
Yating Zhang ◽  
Luqi Zhu ◽  
Pingang He ◽  
Qingjiang Wang
Keyword(s):  
Escherichia Coli ◽  
Capillary Electrophoresis ◽  
High Sensitivity ◽  
Laser Induced Fluorescence ◽  
Galactosidase Activity ◽  
Microchip Capillary Electrophoresis ◽  
Sample Injection ◽  
E Coli ◽  
High Sensitivity Detection ◽  
Sensitivity Detection

A sensitive strategy developed for the detection of Escherichia coli (E. coli) by microchip capillary electrophoresis (MCE) combined with laser-induced fluorescence (LIF) is described in this paper.

scholarly journals Enzyme Characteristics of β-d-Galactosidase- and β-d-Glucuronidase-Positive Bacteria and Their Interference in Rapid Methods for Detection of Waterborne Coliforms andEscherichia coli

1998 ◽  
Vol 64 (3) ◽  
pp. 1018-1023 ◽  
Author(s):  
I. Tryland ◽  
L. Fiksdal
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Enzymatic Activity ◽  
Environmental Water ◽  
Coliform Bacteria ◽  
Galactosidase Activity ◽  
Fluorogenic Substrates ◽  
Rapid Methods ◽  
E Coli ◽  
Aerococcus Viridans

ABSTRACT Bacteria which were β-d-galactosidase and β-d-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-β-d-galactoside and 4-methylumbelliferyl-β-d-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated. The potential interference of false-positive bacteria was evaluated. Several of the β-d-galactosidase-positive nontarget bacteria but none of the β-d-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5°C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different β-d-galactosidase- and β-d-glucuronidase-positive bacteria. The induced and noninduced β-d-glucuronidase activities ofBacillus spp. and Aerococcus viridans were approximately the same as the activities of induced E. coli. Except for some isolates identified asAeromonas spp., all of the induced and noninduced β-d-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean β-d-galactosidase activity than induced E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria.

scholarly journals Activation of the gab Operon in an RpoS-Dependent Manner by Mutations That Truncate the Inner Core of Lipopolysaccharide in Escherichia coli

2004 ◽  
Vol 186 (24) ◽  
pp. 8542-8546 ◽  
Author(s):  
Moses L. Joloba ◽  
Katy M. Clemmer ◽  
Darren D. Sledjeski ◽  
Philip N. Rather
Keyword(s):  
Escherichia Coli ◽  
Sigma Factor ◽  
Inner Core ◽  
Acid Synthesis ◽  
Dependent Manner ◽  
New Genes ◽  
Colanic Acid ◽  
Operon Regulation ◽  
Operon Expression ◽  
High Level

ABSTRACT The gab operon (gabDTPC) in Escherichia coli functions in the conversion of γ-aminobutyrate to succinate. One component of gab operon regulation involves the RpoS sigma factor, which mediates activation at high cell density. Transposon mutagenesis was used to identify new genes that regulate gab operon expression in rich media. A Tn5tmp insertion in the hldD (formerly rfaD) gene increased gabT::lacZ expression 12-fold. The hldD gene product, an ADP-l-glycerol-d-mannoheptose-6-epimerase, catalyzes the conversion of ADP-d-glycerol-d-mannoheptose to ADP-l-glycerol-d-mannoheptose, a precursor for the synthesis of inner-core lipopolysaccharide (LPS). Defined mutations in hldE, required for heptose synthesis, and waaF, required for the addition of the second heptose to the inner core, also resulted in high-level gabT::lacZ expression. The hldD, hldE, and waaF mutants exhibited a mucoid colony phenotype due to production of a colanic acid capsule. However, in the hldD::cat background, the high-level expression of gabT::lacZ was independent of the regulatory components for colanic acid synthesis (rcsA, rcsB, and rcsC) and also independent of manC (cpsB), a structural gene for colanic acid synthesis. Activation of gabT::lacZ in the hldD::cat background was dependent on the RpoS sigma factor. The hldD::cat mutation resulted in a sixfold increase in the levels of a translational RpoS-LacZ fusion and had a marginal effect on a transcriptional fusion. This study reveals a stress-induced pathway, mediated by loss of the LPS inner core, that increases RpoS translation and gab operon expression in E. coli.

scholarly journals Genetic Analysis of Disulfide Isomerization in Escherichia coli: Expression of DsbC Is Modulated by RNase E-Dependent mRNA Processing

2004 ◽  
Vol 186 (3) ◽  
pp. 654-660 ◽  
Author(s):  
Xiaoming Zhan ◽  
Junjun Gao ◽  
Chaitanya Jain ◽  
Michael J. Cieslewicz ◽  
James R. Swartz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Disulfide Bonds ◽  
Critical Role ◽  
Chemical Mutagenesis ◽  
Selection Strategy ◽  
Rnase E ◽  
Disulfide Isomerase ◽  
Isomerase Activity ◽  
Disulfide Isomerization ◽  
Disulfide Isomerase Activity

ABSTRACT We designed a selection strategy for the isolation of Escherichia coli mutants exhibiting enhanced protein disulfide isomerase activity. The folding of a variant of tissue plasminogen activator (v-tPA), a protein containing nine disulfide bonds, in the bacterial periplasm is completely dependent on the level of disulfide isomerase activity of the cell. Mutations that increase this activity mediate the formation of catalytically active v-tPA, which in turn cleaves a p-aminobenzoic acid (PABA)-peptide adduct to release free PABA and thus allows the growth of an auxotrophic strain. Following chemical mutagenesis, a total of eight E. coli mutants exhibiting significantly higher disulfide isomerization activity, not only with v-tPA but also with two other unrelated protein substrates, were isolated. This phenotype resulted from significantly increased expression of the bacterial disulfide isomerase DsbC. In seven of the eight mutants, the upregulation of DsbC was found to be related to defects in RNA processing by RNase E, the rne gene product. Specifically, the genetic lesions in five mutants were shown to be allelic to rne, while an additional two mutants exhibited impaired RNase E activity due to lesions in other loci. The importance of mRNA stability on the expression of DsbC is underscored by the short half-life of the dsbC transcript, which was found to be only 0.8 min at 37°C in wild-type cells but was two- to threefold longer in some of the stronger mutants. These results (i) confirm the central role of DsbC in disulfide bond isomerization in the bacterial periplasm and (ii) suggest a critical role for RNase E in regulating DsbC expression.

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