scholarly journals Dominant negative mutator mutations in the mutS gene of Escherichia coli.

1994 ◽  
Vol 176 (17) ◽  
pp. 5393-5400 ◽  
Author(s):  
T H Wu ◽  
M G Marinus
Keyword(s):  
Escherichia Coli ◽  
Dominant Negative ◽  
Muts Gene

scholarly journals Mutational Analysis of the Locus of Enterocyte Effacement-Encoded Regulator (Ler) of Enteropathogenic Escherichia coli

2008 ◽  
Vol 190 (23) ◽  
pp. 7808-7818 ◽  
Author(s):  
Gal Yerushalmi ◽  
Chen Nadler ◽  
Tatiana Berdichevski ◽  
Ilan Rosenshine
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Mutational Analysis ◽  
Bacterial Colonization ◽  
Point Mutations ◽  
Dominant Negative ◽  
Compensatory Mechanism ◽  
Dependent Manner ◽  
Enterocyte Effacement ◽  
Related Proteins

ABSTRACT The locus of enterocyte effacement (LEE) pathogenicity island of enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) comprises a cluster of operons encoding a type III secretion system and related proteins, all of which are essential for bacterial colonization of the host intestines. The LEE1 operon encodes Ler, which positively regulates many EPEC and EHEC virulence genes located in the LEE region and elsewhere in the chromosome. In addition, Ler is a specific autorepressor of LEE1 transcription. To better understand the function of Ler, we screened for Ler mutants defective in autorepression. We isolated 18 different point mutations in Ler, rendering it defective in autorepression and in DNA binding. Among these mutants were those defective in positive regulation as well as in autorepression, dominant-negative mutants, and a mutant deficient in oligomerization. Importantly, a group of Ler autorepression mutants complemented an EPEC ler deletion mutant for transcription activation in a dosage-dependent manner, suggesting that Ler and possibly other autorepressors have an intrinsic compensatory mechanism that enables them to sustain mutations. In addition, the phenotypes of the different mutants identified by the screen define a novel domain in Ler that is required for oligomerization.

scholarly journals UvrD and UvrD252 Counteract RecQ, RecJ, and RecFOR in a rep Mutant of Escherichia coli

2008 ◽  
Vol 190 (17) ◽  
pp. 5995-6001 ◽  
Author(s):  
Roxane Lestini ◽  
Bénédicte Michel
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Replication Fork ◽  
Synthetic Lethality ◽  
Excision Repair ◽  
Dominant Negative ◽  
Deficient Mutant ◽  
Nucleotide Excision ◽  
Negative Effect ◽  
Presynaptic Proteins

ABSTRACT Rep and UvrD are two related Escherichia coli helicases, and inactivating both is lethal. Based on the observation that the synthetic lethality of rep and uvrD inactivation is suppressed in the absence of the recombination presynaptic proteins RecF, RecO, or RecR, it was proposed that UvrD is essential in the rep mutant to counteract a deleterious RecFOR-dependent RecA binding. We show here that the synthetic lethality of rep and uvrD mutations is also suppressed by recQ and recJ inactivation but not by rarA inactivation. Furthermore, it is independent of the action of UvrD in nucleotide excision repair and mismatch repair. These observations support the idea that UvrD counteracts a deleterious RecA binding to forks blocked in the rep mutant. An ATPase-deficient mutant of UvrD [uvrD(R284A)] is dominant negative in a rep mutant, but only in the presence of all RecQJFOR proteins, suggesting that the UvrD(R284A) mutant protein is deleterious when it counteracts one of these proteins. In contrast, the uvrD252 mutant (G30D), which exhibits a strongly decreased ATPase activity, is viable in a rep mutant, where it allows replication fork reversal. We conclude that the residual ATPase activity of UvrD252 prevents a negative effect on the viability of the rep mutant and allows UvrD to counteract the action of RecQ, RecJ, and RecFOR at forks blocked in the rep mutant. Models for the action of UvrD at blocked forks are proposed.

scholarly journals Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

2002 ◽  
Vol 184 (3) ◽  
pp. 695-705 ◽  
Author(s):  
Joseph C. Chen ◽  
Michael Minev ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Random Mutagenesis ◽  
Dominant Negative ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Negative Effects ◽  
Mutant Proteins ◽  
Division Site

ABSTRACT FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

scholarly journals Enteropathogenic Escherichia coli Activates Ezrin, Which Participates in Disruption of Tight Junction Barrier Function

2001 ◽  
Vol 69 (9) ◽  
pp. 5679-5688 ◽  
Author(s):  
Ivana Simonovic ◽  
Monique Arpin ◽  
Athanasia Koutsouris ◽  
Holly J. Falk-Krzesinski ◽  
Gail Hecht
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Cytoskeletal Proteins ◽  
Dominant Negative ◽  
Intestinal Epithelial ◽  
E Coli ◽  
Membrane Cytoskeleton ◽  
Intestinal Pathogen ◽  
Response Expression

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is an important human intestinal pathogen, especially in infants. EPEC adherence to intestinal epithelial cells induces the accumulation of a number of cytoskeletal proteins beneath the bacteria, including the membrane-cytoskeleton linker ezrin. Evidence suggests that ezrin can participate in signal transduction. The aim of this study was to determine whether ezrin is activated following EPEC infection and if it is involved in the cross talk with host intestinal epithelial cells. We show here that following EPEC attachment to intestinal epithelial cells there was significant phosphorylation of ezrin, first on threonine and later on tyrosine residues. A significant increase in cytoskeleton-associated ezrin occurred following phosphorylation, suggesting activation of this molecule. Nonpathogenic E. coli and EPEC strains harboring mutations in type III secretion failed to elicit this response. Expression of dominant-negative ezrin significantly decreased the EPEC-elicited association of ezrin with the cytoskeleton and attenuated the disruption of intestinal epithelial tight junctions. These results suggest that ezrin is involved in transducing EPEC-initiated signals that ultimately affect host physiological functions.

scholarly journals Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E

2019 ◽  
Vol 48 (2) ◽  
pp. 847-861 ◽  
Author(s):  
Nida Ali ◽  
Jayaraman Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Rna Binding ◽  
Initial Step ◽  
Dominant Negative ◽  
Rnase E ◽  
Wild Type ◽  
Catalytic Active Site

Abstract RNase E is a 472-kDa homo-tetrameric essential endoribonuclease involved in RNA processing and turnover in Escherichia coli. In its N-terminal half (NTH) is the catalytic active site, as also a substrate 5′-sensor pocket that renders enzyme activity maximal on 5′-monophosphorylated RNAs. The protein's non-catalytic C-terminal half (CTH) harbours RNA-binding motifs and serves as scaffold for a multiprotein degradosome complex, but is dispensable for viability. Here, we provide evidence that a full-length hetero-tetramer, composed of a mixture of wild-type and (recessive lethal) active-site mutant subunits, exhibits identical activity in vivo as the wild-type homo-tetramer itself (‘recessive resurrection’). When all of the cognate polypeptides lacked the CTH, the active-site mutant subunits were dominant negative. A pair of C-terminally truncated polypeptides, which were individually inactive because of additional mutations in their active site and 5′-sensor pocket respectively, exhibited catalytic function in combination, both in vivo and in vitro (i.e. intragenic or allelic complementation). Our results indicate that adjacent subunits within an oligomer are separately responsible for 5′-sensing and cleavage, and that RNA binding facilitates oligomerization. We propose also that the CTH mediates a rate-determining initial step for enzyme function, which is likely the binding and channelling of substrate for NTH’s endonucleolytic action.

MUTATOR GENE STUDIES IN ESCHERICHIA COLI: THE mutS GENE

Genetics ◽  
1972 ◽  
Vol 72 (4) ◽  
pp. 551-567 ◽  
Author(s):  
Edward C Cox ◽  
Gerald E Degnen ◽  
Mary L Scheppe
Keyword(s):  
Escherichia Coli ◽  
Lac Operon ◽  
Wild Type ◽  
Mutator Gene ◽  
Sense Strand ◽  
Muts Gene

ABSTRACT We report here on a study of a mutator gene (mutS) that causes transition mutations in Escherichia coli. We have used the trpA system to show that A:T→G:C and G:C→A:T transitions occur. Not all A:T pairs are equally susceptible to mutS action however, since the A:T pair at the trpA223 site reverts at a frequency similar to, if not identical with, the frequency in a mut  + background. Presumably this is a consequence of neighboring bases, because other A:T pairs are reverted by mutS in the same gene; and an A:T pair in the lac operon is reverted at two widely separated points on the chromosome, and in two orientations relative to the trp sense strand. In addition, we have shown that the mutS1 allele is recessive to wild type, and trans active.

scholarly journals The Histidine Kinase Domain of UhpB Inhibits UhpA Action at the Escherichia coli uhpT Promoter

2000 ◽  
Vol 182 (22) ◽  
pp. 6279-6286 ◽  
Author(s):  
Jesse S. Wright ◽  
Igor N. Olekhnovich ◽  
Gail Touchie ◽  
Robert J. Kadner
Keyword(s):  
Escherichia Coli ◽  
Histidine Kinase ◽  
Response Regulator ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Signaling Complex ◽  
Regulatory Systems ◽  
Negative Effect ◽  
Polytopic Membrane Protein

ABSTRACT The histidine kinase (HK) component of many two-component regulatory systems exhibits regulated ability to phosphorylate itself and to participate in transfer of phosphate to and from its cognate response regulator. The signaling system that controls expression of the UhpT sugar phosphate transporter in Escherichia coli in response to external glucose 6-phosphate includes the HK protein UhpB and the polytopic membrane protein UhpC, a UhpT homolog which is required for responsiveness to an inducer and activation of UhpB. The existence of a UhpBC signaling complex is suggested by the requirement for UhpC for the activity of certain constitutively active variants of UhpB, the dominance and epistasis relationships of uhpalleles, and the finding that expression of UhpB in excess of UhpC has a strong dominant-negative effect. Expression of a hybrid protein containing the cytoplasmic C-terminal half of UhpB fused to glutathioneS-transferase (GST) also interfered with Uhp signaling. This interference phenotype could not result solely from the phosphatase activity of UhpB, because interference affected both overexpressed UhpA and UhpA variants which are active in the absence of phosphorylation. Variant forms of UhpB which were active in the absence of UhpC carried amino acid substitutions near motifs conserved in HK proteins. The GST fusion protein inhibited the ability of UhpA to bind and activate transcription at the uhpT promoter. Unlike the wild-type situation, a GST fusion variant carrying one of the UhpB-activating substitutions, R324C, displayed autokinase activity and phosphate transfer to UhpA but retained the ability to sequester UhpA when it was altered in the conserved residues important for phosphate transfer. Thus, the default state of UhpB is kinase off, and activation of its phosphate transfer activity requires either the action of UhpC or the occurrence of certain mutations in UhpB. The interference phenotype shown by UhpB in excess of UhpC appears to include the binding and sequestration of UhpA.

Cloning, expression, and purification of the recombinant pro-apoptotic dominant-negative survivin T34A-C84A protein in Escherichia coli

2019 ◽  
Vol 160 ◽  
pp. 73-83 ◽  
Author(s):  
Shing-Ling Tsai ◽  
Yung-Chieh Chang ◽  
Sailu Sarvagalla ◽  
Shuying Wang ◽  
Mohane Selvaraj Coumar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dominant Negative ◽  
Expression And Purification

scholarly journals Overproduction of a Dominant Mutant of the Conserved Era GTPase Inhibits Cell Division in Escherichia coli

2020 ◽  
Vol 202 (21) ◽  
Author(s):  
Xiaomei Zhou ◽  
Howard K. Peters ◽  
Xintian Li ◽  
Nina Costantino ◽  
Vandana Kumari ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Ribosome Biogenesis ◽  
Cell Mass ◽  
Ribosomal Subunit ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Content Type ◽  
Z Ring

ABSTRACT Cell growth and division are coordinated, ensuring homeostasis under any given growth condition, with division occurring as cell mass doubles. The signals and controlling circuit(s) between growth and division are not well understood; however, it is known in Escherichia coli that the essential GTPase Era, which is growth rate regulated, coordinates the two functions and may be a checkpoint regulator of both. We have isolated a mutant of Era that separates its effect on growth and division. When overproduced, the mutant protein Era647 is dominant to wild-type Era and blocks division, causing cells to filament. Multicopy suppressors that prevent the filamentation phenotype of Era647 either increase the expression of FtsZ or decrease the expression of the Era647 protein. Excess Era647 induces complete delocalization of Z rings, providing an explanation for why Era647 induces filamentation, but this effect is probably not due to direct interaction between Era647 and FtsZ. The hypermorphic ftsZ* allele at the native locus can suppress the effects of Era647 overproduction, indicating that extra FtsZ is not required for the suppression, but another hypermorphic allele that accelerates cell division through periplasmic signaling, ftsL*, cannot. Together, these results suggest that Era647 blocks cell division by destabilizing the Z ring. IMPORTANCE All cells need to coordinate their growth and division, and small GTPases that are conserved throughout life play a key role in this regulation. One of these, Era, provides an essential function in the assembly of the 30S ribosomal subunit in Escherichia coli, but its role in regulating E. coli cell division is much less well understood. Here, we characterize a novel dominant negative mutant of Era (Era647) that uncouples these two activities when overproduced; it inhibits cell division by disrupting assembly of the Z ring, without significantly affecting ribosome production. The unique properties of this mutant should help to elucidate how Era regulates cell division and coordinates this process with ribosome biogenesis.

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