O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 534-537
Author(s):  
S Mitra ◽  
B C Pal ◽  
R S Foote
Keyword(s):  
Escherichia Coli ◽  
Dna Methyltransferase ◽  
Wild Type ◽  
E Coli ◽  
Methylguanine Dna Methyltransferase ◽  
Synthetic Dna ◽  
In The Wild ◽  
Low Levels ◽  
Enzyme Molecules ◽  
Dna Substrate

O(6)-Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3H-labeled O(6)-methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase.

scholarly journals Global Role for ClpP-Containing Proteases in Stationary-Phase Adaptation of Escherichia coli

2003 ◽  
Vol 185 (1) ◽  
pp. 115-125 ◽  
Author(s):  
Dieter Weichart ◽  
Nadine Querfurth ◽  
Mathias Dreger ◽  
Regine Hengge-Aronis
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Growth Phase ◽  
Proteome Analysis ◽  
Resting Cells ◽  
Wild Type ◽  
Late Stationary Phase ◽  
E Coli ◽  
In The Wild ◽  
Dps Protein

ABSTRACT To elucidate the involvement of proteolysis in the regulation of stationary-phase adaptation, the clpA, clpX, and clpP protease mutants of Escherichia coli were subjected to proteome analysis during growth and during carbon starvation. For most of the growth-phase-regulated proteins detected on our gels, the clpA, clpX, or clpP mutant failed to mount the growth-phase regulation found in the wild type. For example, in the clpP and clpA mutant cultures, the Dps protein, the WrbA protein, and the periplasmic lysine-arginine-ornithine binding protein ArgT did not display the induction typical for late-stationary-phase wild-type cells. On the other hand, in the protease mutants, a number of proteins accumulated to a higher degree than in the wild type, especially in late stationary phase. The proteins affected in this manner include the LeuA, TrxB, GdhA, GlnA, and MetK proteins and alkyl hydroperoxide reductase (AhpC). These proteins may be directly degraded by ClpAP or ClpXP, respectively, or their expression could be modulated by a protease-dependent mechanism. From our data we conclude that the levels of most major growth-phase-regulated proteins in E. coli are at some point controlled by the activity of at least one of the ClpP, ClpA, and ClpX proteins. Cultures of the strains lacking functional ClpP or ClpX also displayed a more rapid loss of viability during extended stationary phase than the wild type. Therefore, regulation by proteolysis seems to be more important, especially in resting cells, than previously suspected.

Isolation and characterization of catabolite repression-resistant mutants of Escherichia coli

1977 ◽  
Vol 23 (10) ◽  
pp. 1384-1393 ◽  
Author(s):  
Glen D. Armstrong ◽  
Hiroshi Yamazaki
Keyword(s):  
Escherichia Coli ◽  
Catabolite Repression ◽  
Coli Strain ◽  
Wild Type ◽  
Phosphotransferase System ◽  
E Coli ◽  
Isolation And Characterization ◽  
In The Wild ◽  
Resistant Mutants

A method has been developed for the isolation of Escherichia coli mutants which are resistant to catabolite repression. The method is based on the fact that a mixture of glucose and gluconate inhibits the development of chemotactic motility in the wild type, but not in the mutants. A motile E. coli strain was mutagenized and grown in glucose and gluconate. Mutants which were able to swim into a tube containing a chemotactic attractant (aspartic acid) were isolated. Most of these mutants were able to produce β-galactosidase in the presence of glucose and gluconate and were normal in their ability to degrade adenosine 3′,5′-cyclic monophosphate. Some of these mutants were defective in the glucose phosphotransferase system.

scholarly journals Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli

1982 ◽  
Vol 152 (3) ◽  
pp. 1241-1247
Author(s):  
H Berger ◽  
J Hacker ◽  
A Juarez ◽  
C Hughes ◽  
W Goebel
Keyword(s):  
Escherichia Coli ◽  
High Frequency ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Genetic Determinants ◽  
E Coli ◽  
Gene Banks ◽  
In The Wild ◽  
K 12 ◽  
Type Strains

We have cloned the chromosomal hemolysin determinants from Escherichia coli strains belonging to the four O-serotypes O4, O6, O18, and O75. The hemolysin-producing clones were isolated from gene banks of these strains which were constructed by inserting partial Sau3A fragments of chromosomal DNA into the cosmid pJC74. The hemolytic cosmid clones were relatively stable. The inserts were further subcloned either as SalI fragments in pACYC184 or as BamHI-SalI fragments in a recombinant plasmid (pANN202) containing cistron C (hlyC) of the plasmid-encoded hemolysin determinant. Detailed restriction maps of each of these determinants were constructed, and it was found that, despite sharing overall homology, the determinants exhibited minor specific differences in their structure. These appeared to be restricted to cistron A (hlyA), which is the structural gene for hemolysin. In the gene banks of two of these hemolytic strains, we could also identify clones which carried the genetic determinants for the mannose-resistant hemagglutination antigens Vb and VIc. Both of these fimbrial antigens were expressed in the E. coli K-12 clones to an extent similar to that observed in the wild-type strains. These recombinant cosmids were rather unstable, and, in the absence of selection, segregated at a high frequency.

scholarly journals Function of the ςE Regulon in Dead-Cell Lysis in Stationary-Phase Escherichia coli

2000 ◽  
Vol 182 (18) ◽  
pp. 5231-5237 ◽  
Author(s):  
Takeshi Nitta ◽  
Hiroshi Nagamitsu ◽  
Masayuki Murata ◽  
Hanae Izu ◽  
Mamoru Yamada
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Cell Lysis ◽  
Dead Cell ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
E Coli ◽  
Intracellular Proteins ◽  
In The Wild ◽  
Cloned Gene

ABSTRACT Elevation of active ςE levels in Escherichia coli by either repressing the expression of rseAencoding an anti-ςE factor or cloning rpoE in a multicopy plasmid, led to a large decrease in the number of dead cells and the accumulation of cellular proteins in the medium in the stationary phase. The numbers of CFU, however, were nearly the same as those of the wild type or cells devoid of the cloned gene. In the wild-type cells, rpoE expression was increased in the stationary phase and a low-level release of intracellular proteins was observed. These results suggest that dead cell lysis in stationary-phase E. coli occurs in a ςE-dependent fashion. We propose there is a novel physiological function of the ςE regulon that may guarantee cell survival in prolonged stationary phase by providing nutrients from dead cells for the next generation.

scholarly journals Analysis of the Function of Enteropathogenic Escherichia coli EspB by Random Mutagenesis

2006 ◽  
Vol 74 (2) ◽  
pp. 810-820 ◽  
Author(s):  
Wensheng Luo ◽  
Michael S. Donnenberg
Keyword(s):  
Escherichia Coli ◽  
Hemolytic Activity ◽  
Deletion Mutant ◽  
Protein Translocation ◽  
Random Mutagenesis ◽  
Wild Type ◽  
Filamentous Actin ◽  
Infantile Diarrhea ◽  
E Coli ◽  
Low Levels

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is an important cause of infantile diarrhea, especially in developing countries. EspB, a key virulence factor of EPEC, is required for the attaching and effacing effect characteristic of EPEC and enterohemorrhagic E. coli and has been posited to play several functions in the process of infection. Attaching and effacing activity is associated with the accumulation of filamentous actin beneath adherent bacteria as measured in the fluorescence actin staining (FAS) test. To determine whether different domains of EspB are responsible for different functions, 42 plasmids carrying mutated espB were introduced into an espB deletion mutant. Two major groups of espB mutants were identified. One group of 17 mutants exhibited positive FAS results and normal levels of hemolytic activity. Another group of 22 mutants exhibited negative FAS results and low levels of hemolytic activity. Three mutants were exceptional. One mutant was FAS positive but had significantly reduced hemolytic activity. Conversely, a second mutant was FAS negative but had full hemolytic activity. A third mutant had a significantly reduced FAS level compared to the wild type but full hemolytic activity. The results of EspF and Tir translocation assays confirmed that FAS-negative insertions disrupt effector translocation and mutants with FAS-positive insertions retain protein translocation activity. These results suggest that EspB has distinct domain functions involved in effector translocation that can be distinguished from its role as a component of the translocation pore.

scholarly journals UDP-N-Acetylmuramic Acid l-Alanine Ligase (MurC) Inhibition in atolCMutant Escherichia coli Strain Leads to Cell Death

2014 ◽  
Vol 58 (10) ◽  
pp. 6165-6171 ◽  
Author(s):  
Vaishali Humnabadkar ◽  
K. R. Prabhakar ◽  
Ashwini Narayan ◽  
Sreevalli Sharma ◽  
Supreeth Guptha ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Coli Strain ◽  
Cellular Activity ◽  
Wild Type ◽  
Compound A ◽  
Content Type ◽  
E Coli ◽  
In The Wild ◽  
High Concentrations

ABSTRACTThe Mur ligases play an essential role in the biosynthesis of bacterial peptidoglycan and hence are attractive antibacterial targets. A screen of the AstraZeneca compound library led to the identification of compound A, a pyrazolopyrimidine, as a potent inhibitor ofEscherichia coliandPseudomonas aeruginosaMurC. However, cellular activity againstE. coliorP. aeruginosawas not observed. Compound A was active against efflux pump mutants of both strains. Experiments using anE. colitolCmutant revealed accumulation of the MurC substrate and a decrease in the level of product upon treatment with compound A,indicating inhibition of MurC enzyme in these cells. Such a modulation was not observed in theE. coliwild-type cells. Further, overexpression of MurC in theE. colitolCmutant led to an increase in the compound A MIC by ≥16-fold, establishing a correlation between MurC inhibition and cellular activity. In addition, estimation of the intracellular compound A level showed an accumulation of the compound over time in thetolCmutant strain. A significant compound A level was not detected in the wild-typeE. colistrain even upon treatment with high concentrations of the compound. Therefore, the lack of MIC and absence of MurC inhibition in wild-typeE. coliwere possibly due to suboptimal compound concentration as a consequence of a high efflux level and/or poor permeativity of compound A.

scholarly journals Expression of Hemin Receptor Molecule ChuA Is Influenced by RfaH in Uropathogenic Escherichia coliStrain 536

2001 ◽  
Vol 69 (3) ◽  
pp. 1924-1928 ◽  
Author(s):  
Gábor Nagy ◽  
Ulrich Dobrindt ◽  
Maren Kupfer ◽  
Levente Emödy ◽  
Helge Karch ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Protein ◽  
Wild Type ◽  
Receptor Molecule ◽  
E Coli ◽  
Protein Levels ◽  
In The Wild

ABSTRACT The outer membrane protein ChuA responsible for hemin utilization has been recently identified in several pathogenic Escherichia coli strains. We report that the regulatory protein RfaH influences ChuA expression in the uropathogenic E. colistrain 536. In an rfaH mutant, the chuAtranscript as well as the ChuA protein levels were significantly decreased in comparison with those in the wild-type strain. Within thechuA gene, a consensus motif known as the JUMPStart (just upstream of many polysaccharide associated gene starts) sequence was found, which is shared by RfaH-affected operons. Furthermore, the presence of two different subclasses of thechuA determinant and their distribution in E. coli pathogroups are described.

scholarly journals Biochemical Genetics of the Cryptic Gene System for Cellobiose Utilization in Escherichia coli K12

Genetics ◽  
1987 ◽  
Vol 115 (3) ◽  
pp. 419-429
Author(s):  
Maja Kricker ◽  
Barry G Hall
Keyword(s):  
Escherichia Coli ◽  
Microbial Evolution ◽  
Transport Protein ◽  
Wild Type ◽  
Phosphotransferase System ◽  
E Coli ◽  
Escherichia Coli K12 ◽  
Biochemical Assays ◽  
In The Wild

ABSTRACT The cellobiose catabolic system of Escherichia coli K12 is being used to study the role of cryptic genes in microbial evolution. Wild-type E. coli K12 do not utilize the β-glucoside sugars, arbutin, salicin and cellobiose. A Cel+ (cellobiose utilizing) mutant which grows on cellobiose, arbutin, and salicin was isolated previously from wild-type E. coli K12. Biochemical assays indicate that a cel structural gene (celT) specifies a single transport protein that is a β-glucoside specific enzyme of the phosphoenolpyruvate-dependent phosphotransferase system. The transport protein phosphorylates β-glucosides at the expense of phosphoenolpyruvate. A single phosphoglucosidase, specified by celH, hydrolyzes phosphorylated cellobiose, arbutin, and salicin. The genes of the cel system are expressed constitutively in the Cel+ mutant, whereas they are not expressed at a detectable level in the wild-type strain. The transport and hydrolase genes are simultaneously silenced or simultaneously expressed and thus constitute an operon. Cel+ strains which fail to utilize one or more β-glucosides express the transport system at a lower level than do Cel+ strains which grow on all three β-glucosides. Other strains inducibly express a gene which specifies transport of arbutin but not the other β-glucosides. The arbutin transport gene, arbT, maps outside of the cel locus.

scholarly journals Inducible l-Alanine Exporter Encoded by the Novel GeneygaW(alaE) in Escherichia coli

2011 ◽  
Vol 77 (12) ◽  
pp. 4027-4034 ◽  
Author(s):  
Hatsuhiro Hori ◽  
Hiroshi Yoneyama ◽  
Ryuta Tobe ◽  
Tasuke Ando ◽  
Emiko Isogai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Wild Type Strain ◽  
Chemical Mutagenesis ◽  
The Novel ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Export Rate ◽  
In The Wild ◽  
Export System

ABSTRACTWe previously isolated a mutant hypersensitive tol-alanyl-l-alanine from a non-l-alanine-metabolizingEscherichia colistrain and found that it lacked an induciblel-alanine export system. Consequently, this mutant showed a significant accumulation of intracellularl-alanine and a reduction in thel-alanine export rate compared to the parent strain. When the mutant was used as a host to clone a gene(s) that complements the dipeptide-hypersensitive phenotype, two uncharacterized genes,ygaWandytfF, and two characterized genes,yddGandyeaS, were identified. Overexpression of each gene in the mutant resulted in a decrease in the intracellularl-alanine level and enhancement of thel-alanine export rate in the presence of the dipeptide, suggesting that their products function as exporters ofl-alanine. SinceygaWexhibited the most striking impact on both the intra- and the extracellularl-alanine levels among the four genes identified, we disrupted theygaWgene in the non-l-alanine-metabolizing strain. The resulting isogenic mutant showed the same intra- and extracellularl-alanine levels as observed in the dipeptide-hypersensitive mutant obtained by chemical mutagenesis. When each gene was overexpressed in the wild-type strain, which does not intrinsically excrete alanine, only theygaWgene conferred on the cells the ability to excrete alanine. In addition, expression of theygaWgene was induced in the presence of the dipeptide. On the basis of these results, we concluded that YgaW is likely to be the physiologically most relevant exporter forl-alanine inE. coliand proposed that the gene be redesignatedalaEforalanineexport.

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