Cloning, sequencing, and expression of cscA invertase from Escherichia coli B-62

1999 ◽  
Vol 45 (5) ◽  
pp. 418-422 ◽  
Author(s):  
Miklós Sahin-Tóth ◽  
Zsolt Lengyel ◽  
Hiroshi Tsunekawa
Keyword(s):  
Escherichia Coli ◽  
Functional Characterization ◽  
Reading Frame ◽  
E Coli ◽  
Invertase Gene ◽  
Sequencing And Expression ◽  
High Level ◽  
K 12 ◽  
Sucrose Utilization ◽  
Escherichia Coli B

We have isolated a 2.5-kb DNA fragment from plasmid pST5R7 encoding a sucrose utilization system from Escherichia coli B-62 which confers a sucrose-fermenting phenotype to transformed E. coli K-12 strains. DNA-sequence determination revealed one full-length open reading frame 98% identical to cscA, the sucrose-hydrolase (invertase) gene of the csc regulon from E. coli EC3132. Functional characterization indicates that high-level expression and limited periplasmic release of invertase is responsible for the sucrose-fermenting capacity of transformed E. coli K-12 strains carrying cscA.Key words: sucrose utilization, sucrose hydrolase, invertase, recombinant protein production.

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

scholarly journals Molecular Control of Sucrose Utilization in Escherichia coli W, an Efficient Sucrose-Utilizing Strain

2012 ◽  
Vol 79 (2) ◽  
pp. 478-487 ◽  
Author(s):  
Suriana Sabri ◽  
Lars K. Nielsen ◽  
Claudia E. Vickers
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Good Growth ◽  
Knockout Mutant ◽  
Sucrose Transport ◽  
Molecular Control ◽  
Content Type ◽  
E Coli ◽  
K 12 ◽  
Sucrose Utilization

ABSTRACTSucrose is an industrially important carbon source for microbial fermentation. Sucrose utilization inEscherichia coli, however, is poorly understood, and most industrial strains cannot utilize sucrose. The roles of the chromosomally encoded sucrose catabolism (csc) genes inE. coliW were examined by knockout and overexpression experiments. At low sucrose concentrations, thecscgenes are repressed and cells cannot grow. Removal of either the repressor protein (cscR) or the fructokinase (cscK) gene facilitated derepression. Furthermore, combinatorial knockout ofcscRandcscKconferred an improved growth rate on low sucrose. The invertase (cscA) and sucrose transporter (cscB) genes are essential for sucrose catabolism inE. coliW, demonstrating that no other genes can provide sucrose transport or inversion activities. However,cscKis not essential for sucrose utilization. Fructose is excreted into the medium by thecscK-knockout strain in the presence of high sucrose, whereas at low sucrose (when carbon availability is limiting), fructose is utilized by the cell. Overexpression ofcscA,cscAK, orcscABcould complement the WΔcscRKABknockout mutant or confer growth on a K-12 strain which could not naturally utilize sucrose. However, phenotypic stability and relatively good growth rates were observed in the K-12 strain only when overexpressingcscAB, and full growth rate complementation in WΔcscRKABalso requiredcscAB. Our understanding of sucrose utilization can be used to improveE. coliW and engineer sucrose utilization in strains which do not naturally utilize sucrose, allowing substitution of sucrose for other, less desirable carbon sources in industrial fermentations.

Understanding the Differences between Genome Sequences of Escherichia coli B Strains REL606 and BL21(DE3) and Comparison of the E. coli B and K-12 Genomes

2009 ◽  
Vol 394 (4) ◽  
pp. 653-680 ◽  
Author(s):  
F. William Studier ◽  
Patrick Daegelen ◽  
Richard E. Lenski ◽  
Sergei Maslov ◽  
Jihyun F. Kim
Keyword(s):  
Escherichia Coli ◽  
Genome Sequences ◽  
E Coli ◽  
K 12 ◽  
Escherichia Coli B

scholarly journals Rhs Elements Comprise Three Subfamilies Which Diverged Prior to Acquisition by Escherichia coli

1998 ◽  
Vol 180 (16) ◽  
pp. 4102-4110 ◽  
Author(s):  
Yong-Dong Wang ◽  
Sheng Zhao ◽  
Charles W. Hill
Keyword(s):  
Escherichia Coli ◽  
Phylogenetic Analysis ◽  
Open Reading Frame ◽  
Large Protein ◽  
Rich Region ◽  
Reading Frame ◽  
E Coli ◽  
The Core ◽  
Reference Collection ◽  
K 12

ABSTRACT The Rhs elements are complex genetic composites widely spread among Escherichia coli isolates. One of their components, a 3.7-kb, GC-rich core, maintains a single open reading frame that extends the full length of the core and then 400 to 600 bp beyond into an AT-rich region. Whereas Rhs cores are homologous, core extensions from different elements are dissimilar. Two new Rhs elements from strains of the ECOR reference collection have been characterized. RhsG (from strain ECOR-11) maps to min 5.3, and RhsH (from strain ECOR-45) maps to min 32.8, where it lies in tandem with RhsE. Comparison of strain K-12 to ECOR-11 indicates that RhsGwas once present in but has been largely deleted from an ancestor of K-12. Phylogenetic analysis shows that the cores from eight known elements fall into three subfamilies, RhsA-B-C-F,RhsD-E, and RhsG-H. Cores from different subfamilies diverge 22 to 29%. Analysis of substitutions that distinguish between subfamilies shows that the origin of the ancestral core as well as the process of subfamily separation occurred in a GC-rich background. Furthermore, each subfamily independently passed from the GC-rich background to a less GC-rich background such asE. coli. A new example of core-extension shuffling provides the first example of exchange between cores of different subfamilies. A novel component of RhsE and RhsG,vgr, encodes a large protein distinguished by 18 to 19 repetitions of a Val-Gly dipeptide occurring with a eight-residue periodicity.

scholarly journals Correlation of Rhs elements with Escherichia coli population structure.

Genetics ◽  
1995 ◽  
Vol 141 (1) ◽  
pp. 15-24
Author(s):  
C W Hill ◽  
G Feulner ◽  
M S Brody ◽  
S Zhao ◽  
A B Sadosky ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Open Reading Frame ◽  
Clonal Structure ◽  
Reading Frame ◽  
E Coli ◽  
Sequence Identity ◽  
Reference Collection ◽  
K 12 ◽  
Recent Variation ◽  
Fourth Member

Abstract The Rhs family of composite genetic elements was assessed for variation among independent Escherichia coli strains of the ECOR reference collection. The location and content of the RhsA-B-C-F subfamily correlates highly with the clonal structure of the ECOR collection. This correlation exists at several levels: the presence of Rhs core homology in the strain, the location of the Rhs elements present, and the identity of the Rhs core-extensions associated with each element. A provocative finding was that an identical 1518-bp segment, covering core-extension-b1 and its associated downstream open reading frame, is present in two distinct clonal groups, but in association with different Rhs elements. The sequence identity of this segment when contrasted with the divergence of other chromosomal segments suggests that shuffling of Rhs core extensions has been a relatively recent variation. Nevertheless the copies of core-extension-b1 were placed within the respective Rhs elements before the emergence of the clonal groups. In the course of this analysis, two new Rhs elements absent from E. coli K-12 were discovered: RhsF, a fourth member of the RhsA-B-C-F subfamily, and RhsG, the prototype of a third Rhs subfamily.

Compilation of Escherichia coli K-12 outer membrane phage receptors – their function and some historical remarks

2020 ◽  
Vol 367 (2) ◽  
Author(s):  
Klaus Hantke
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Functional Characterization ◽  
Historical Context ◽  
E Coli ◽  
Great Progress ◽  
Historical Remarks ◽  
K 12 ◽  
Made In

ABSTRACT Many Escherichia coli phages have been sequenced, but in most cases their sequences alone do not suffice to predict their host specificity. Analysis of phage resistant E. coli K-12 mutants have uncovered a certain set of outer membrane proteins and polysaccharides as receptors. In this review, a compilation of E. coli K12 phage receptors is provided and their functional characterization, often driven by studies on phage resistant mutants, is discussed in the historical context. While great progress has been made in this field thus far, several proteins in the outer membrane still await characterization as phage receptors.

scholarly journals Characterization of the Novel Factor Paa Involved in the Early Steps of the Adhesion Mechanism of Attaching and Effacing Escherichia coli

2003 ◽  
Vol 71 (8) ◽  
pp. 4516-4525 ◽  
Author(s):  
Isabelle Batisson ◽  
Marie-Pierre Guimond ◽  
Francis Girard ◽  
Hongyan An ◽  
Chengru Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Insertion Mutagenesis ◽  
Intestinal Epithelial ◽  
The Novel ◽  
Reading Frame ◽  
E Coli ◽  
Transposon Insertion ◽  
Gene Sequence Analysis ◽  
Highly Correlated ◽  
K 12

ABSTRACT Nonenterotoxigenic porcine Escherichia coli strains belonging to the serogroup O45 have been associated with postweaning diarrhea in swine and adhere to intestinal epithelial cells in a characteristic attaching and effacing (A/E) pattern. O45 porcine enteropathogenic E. coli (PEPEC) strain 86-1390 induces typical A/E lesions in a pig ileal explant model. Using TnphoA transposon insertion mutagenesis on strain 86-1390, we found a mutant that did not induce A/E lesions. The insertion was identified in a gene designated paa (porcine A/E-associated gene). Sequence analysis of paa revealed an open reading frame of 753 bp encoding a 27.6-kDa protein which displayed 100, 51.8, and 49% homology with Paa of enterohemorrhagic E. coli O157:H7 strains (EDL933 and Sakai), PEB3 of Campylobacter jejuni, and AcfC of Vibrio cholerae, respectively. Chromosomal localization studies indicated that the region containing paa was inserted between the yciD and yciE genes at about 28.3 min of the E. coli K-12 chromosome. The presence of paa and eae sequences in the porcine O45 strains is highly correlated with the A/E phenotype. However, the observation that three eae-positive but paa-negative PEPEC O45 strains were A/E negative provides further evidence for the importance of the paa gene in the A/E activity of O45 strains. As well, the complementation of the paa mutant restored the A/E activity of the 86-1390 strain, showing the involvement of Paa in PEPEC pathogenicity. These observations suggest that Paa contributes to the early stages of A/E E. coli virulence.

scholarly journals Regulation of Aerobic and Anaerobic d-Malate Metabolism of Escherichia coli by the LysR-Type Regulator DmlR (YeaT)

2010 ◽  
Vol 192 (10) ◽  
pp. 2503-2511 ◽  
Author(s):  
Hanna Lukas ◽  
Julia Reimann ◽  
Ok Bin Kim ◽  
Jan Grimpo ◽  
Gottfried Unden
Keyword(s):  
Escherichia Coli ◽  
Malate Dehydrogenase ◽  
Major Effect ◽  
Anaerobic Growth ◽  
Two Component System ◽  
E Coli ◽  
High Level ◽  
K 12 ◽  
Aerobic And Anaerobic ◽  
Malate Metabolism

ABSTRACT Escherichia coli K-12 is able to grow under aerobic conditions on d-malate using DctA for d-malate uptake and the d-malate dehydrogenase DmlA (formerly YeaU) for converting d-malate to pyruvate. Induction of dmlA encoding DmlA required an intact dmlR (formerly yeaT) gene, which encodes DmlR, a LysR-type transcriptional regulator. Induction of dmlA by DmlR required the presence of d-malate or l- or meso-tartrate, but only d-malate supported aerobic growth. The regulator of general C4-dicarboxylate metabolism (DcuS-DcuR two-component system) had some effect on dmlA expression. The anaerobic l-tartrate regulator TtdR or the oxygen sensors ArcB-ArcA and FNR did not have a major effect on dmlA expression. DmlR has a high level of sequence identity (49%) with TtdR, the l- and meso-tartrate-specific regulator of l-tartrate fermentation in E. coli. dmlA was also expressed at high levels under anaerobic conditions, and the bacteria had d-malate dehydrogenase activity. These bacteria, however, were not able to grow on d-malate since the anaerobic pathway for d-malate degradation has a predicted yield of ≤0 ATP/mol d-malate. Slow anaerobic growth on d-malate was observed when glycerol was also provided as an electron donor, and d-malate was used in fumarate respiration. The expression of dmlR is subject to negative autoregulation. The network for regulation and coordination of the central and peripheral pathways for C4-dicarboxylate metabolism by the regulators DcuS-DcuR, DmlR, and TtdR is discussed.

scholarly journals Deficiency in l-Serine Deaminase Interferes with One-Carbon Metabolism and Cell Wall Synthesis in Escherichia coli K-12

2010 ◽  
Vol 192 (20) ◽  
pp. 5515-5525 ◽  
Author(s):  
Xiao Zhang ◽  
Ziad W. El-Hajj ◽  
Elaine Newman
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Division ◽  
Amino Acid ◽  
Cell Wall Synthesis ◽  
E Coli ◽  
Amino Acid Mixtures ◽  
Glycine Cleavage ◽  
High Level ◽  
K 12

ABSTRACT Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes l-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S l-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of l-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C1 units and interferes with cell wall synthesis. We suggest that at high concentrations, l-serine decreases synthesis of UDP-N-acetylmuramate-l-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high l-serine is overcome in several ways: by a large concentration of l-alanine, by overproducing MurC together with a low concentration of l-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide.

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