scholarly journals The MutS C Terminus Is Essential for Mismatch Repair Activity In Vivo

2005 ◽  
Vol 187 (18) ◽  
pp. 6577-6579 ◽  
Author(s):  
Melissa A. Calmann ◽  
Anetta Nowosielska ◽  
M. G. Marinus
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Amino Acids ◽  
Cytotoxic Agents ◽  
Chromosomal Deletion ◽  
C Terminus ◽  
Mutation Avoidance ◽  
Repair Activity ◽  
K 12

ABSTRACT An Escherichia coli K-12 strain was constructed with a chromosomal deletion (mutSΔ800) in the mutS gene that produced the removal of the C-terminal 53 amino acids which are not present in the MutS crystal structure. This strain has a MutS null phenotype for mutation avoidance, antirecombination, and sensitivity to cytotoxic agents in a dam mutant background.

scholarly journals The C-Terminal Flexible Domain of the Heme Chaperone CcmE Is Important but Not Essential for Its Function

2003 ◽  
Vol 185 (13) ◽  
pp. 3821-3827 ◽  
Author(s):  
Elisabeth Enggist ◽  
Linda Thöny-Meyer
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Heme Binding ◽  
Membrane Anchor ◽  
Terminal Domain ◽  
Conserved Domain ◽  
C Terminus ◽  
Helical Turn ◽  
Low Activity

ABSTRACT CcmE is a heme chaperone active in the cytochrome c maturation pathway of Escherichia coli. It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome c. The recently solved structure of soluble CcmE revealed a compact core consisting of a β-barrel and a flexible C-terminal domain with a short α-helical turn. In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus. Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely. For detectable transfer of heme to type c cytochromes, only one additional residue, D131, was required, and for efficient cytochrome c maturation, the seven-residue sequence 131DENYTPP137 was required. When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE. Soluble full-length CcmE had low activity in cytochrome c maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

In vivo genesis of a transposon carrying the histidine genes of Escherichia coli K-12

1984 ◽  
Vol 193 (1) ◽  
pp. 172-178
Author(s):  
S. Palchaudhuri ◽  
T. M. Lakshmi ◽  
M. S. Judge ◽  
J. Murthy
Keyword(s):  
Escherichia Coli ◽  
K 12

scholarly journals Competition between congenic Escherichia coli K-12 strains in vivo.

1981 ◽  
Vol 32 (1) ◽  
pp. 74-79 ◽  
Author(s):  
A Onderdonk ◽  
B Marshall ◽  
R Cisneros ◽  
S B Levy
Keyword(s):  
Escherichia Coli ◽  
K 12

scholarly journals An Epistasis Analysis of recA and recN in Escherichia coli K-12

Genetics ◽  
2020 ◽  
Vol 216 (2) ◽  
pp. 381-393
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Double Mutant ◽  
Strand Break ◽  
Wild Type ◽  
Epistasis Analysis ◽  
Chromatid Cohesion ◽  
K 12 ◽  
Structural Maintenance Of Chromosomes ◽  
Sos Protein

RecA is essential for double-strand-break repair (DSBR) and the SOS response in Escherichia coli K-12. RecN is an SOS protein and a member of the Structural Maintenance of Chromosomes family of proteins thought to play a role in sister chromatid cohesion/interactions during DSBR. Previous studies have shown that a plasmid-encoded recA4190 (Q300R) mutant had a phenotype similar to ∆recN (mitomycin C sensitive and UV resistant). It was hypothesized that RecN and RecA physically interact, and that recA4190 specifically eliminated this interaction. To test this model, an epistasis analysis between recA4190 and ∆recN was performed in wild-type and recBC sbcBC cells. To do this, recA4190 was first transferred to the chromosome. As single mutants, recA4190 and ∆recN were Rec+ as measured by transductional recombination, but were 3-fold and 10-fold decreased in their ability to do I-SceI-induced DSBR, respectively. In both cases, the double mutant had an additive phenotype relative to either single mutant. In the recBC sbcBC background, recA4190 and ∆recN cells were very UVS (sensitive), Rec−, had high basal levels of SOS expression and an altered distribution of RecA-GFP structures. In all cases, the double mutant had additive phenotypes. These data suggest that recA4190 (Q300R) and ∆recN remove functions in genetically distinct pathways important for DNA repair, and that RecA Q300 was not important for an interaction between RecN and RecA in vivo. recA4190 (Q300R) revealed modest phenotypes in a wild-type background and dramatic phenotypes in a recBC sbcBC strain, reflecting greater stringency of RecA’s role in that background.

scholarly journals Identification of Escherichia coli Genes That Are Specifically Expressed in a Murine Model of Septicemic Infection

2002 ◽  
Vol 70 (7) ◽  
pp. 3404-3412 ◽  
Author(s):  
Muhammad A. Khan ◽  
Richard E. Isaacson
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Anaerobic Respiration ◽  
Disease Process ◽  
Antimicrobial Drugs ◽  
Reverse Transcription Pcr ◽  
Successful Infection ◽  
K 12

ABSTRACT Identification and characterization of bacterial genes that are induced during the disease process are important in understanding the molecular mechanism of disease and can be useful in designing antimicrobial drugs to control the disease. The identification of in vivo induced (ivi) genes of an Escherichia coli septicemia strain by using antibiotic-based in vivo expression technology is described. Bacterial clones resistant to chloramphenicol in vivo were recovered from the livers of infected mice. Most of the ivi clones were sensitive to chloramphenicol when grown in vitro. Using reverse transcription-PCR, it was demonstrated that selected ivi clones expressed cat in the livers of infected mice but not during in vitro growth. A total of 750 colonies were recovered after three successive rounds of in vivo selection, and 168 isolated ivi clones were sequenced. The sequence analysis revealed that 37 clones encoded hypothetical proteins found in E. coli K-12, whereas 10 clones contained genes that had no significant homology to DNA sequences in GenBank. Two clones were found to contain transposon-related functions. Other clones contained genes required for amino acid metabolism, anaerobic respiration, DNA repair, the heat shock response, and the cellular repressor of the SOS response. In addition, one clone contained the aerobactin biosynthesis gene iucA. Mutations were introduced in to seven of the identified ivi genes. An in vivo mouse challenge-competition assay was used to determine if the mutants were attenuated. The results suggested that these ivi genes were important for survival in vivo, and three of the seven mutant ivi clones were required for successful infection of mice.

Chromosomal Integration of Heterologous DNA in Escherichia coli with Precise Removal of Markers and Replicons Used during Construction

1999 ◽  
Vol 181 (22) ◽  
pp. 7143-7148 ◽  
Author(s):  
F. Martinez-Morales ◽  
A. C. Borges ◽  
A. Martinez ◽  
K. T. Shanmugam ◽  
L. O. Ingram
Keyword(s):  
Escherichia Coli ◽  
Helper Plasmid ◽  
Flp Recombinase ◽  
E Coli ◽  
Homologous Fragment ◽  
Genes Encoding ◽  
Dna Insertion ◽  
K 12 ◽  
Multiple Cloning Sites

ABSTRACT A set of vectors which facilitates the sequential integration of new functions into the Escherichia coli chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426–4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single FRT site. The modified vectors contain twoFRT sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single FRT site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating Zymomonas mobilis genes encoding the ethanol pathway behind the native chromosomaladhE gene in strains of E. coli K-12 andE. coli B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of E. coli strains with precise deletion of extraneous genes used during construction.

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