An assessment of the role of intracellular free Ca2+ in E. coli

Holland, I. B.; Jones, Helen; Campbell, Anthony K.; Jacq, Annick

doi:10.1016/s0300-9084(99)00205-9

Cited by 88 publications

(31 citation statements)

References 41 publications

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“…Although the importance of Ca 2+ as a regulator in eukaryotes is well-established, its role in prokaryotes is less clear (28). However, it has been implicated in a variety of processes including DNA replication and cell division (29,30); our results raise the possibility of a role for Ca 2+ in regulating the activities of gyrase, a key enzyme in DNA replication.…”

Section: Introductionmentioning

confidence: 58%

The role of Ca 2+ in the activity of Mycobacterium tuberculosis DNA gyrase

Karkare

Yousafzai

Mitchenall

et al. 2012

Nucleic Acids Research

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DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis and needs to catalyse DNA supercoiling, relaxation and decatenation reactions in order to fulfil the functions normally carried out by gyrase and DNA topoisomerase IV in other bacteria. We have obtained evidence for the existence of a Ca2+-binding site in the GyrA subunit of M. tuberculosis gyrase. Ca2+ cannot support topoisomerase reactions in the absence of Mg2+, but partial removal of Ca2+ from GyrA by dialysis against EGTA leads to a modest loss in relaxation activity that can be restored by adding back Ca2+. More extensive removal of Ca2+ by denaturation of GyrA and dialysis against EGTA results in an enzyme with greatly reduced enzyme activities. Mutation of the proposed Ca2+-binding residues also leads to loss of activity. We propose that Ca2+ has a regulatory role in M. tuberculosis gyrase and suggest a model for the modulation of gyrase activity by Ca2+ binding.

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Section: Introductionmentioning

confidence: 58%

The role of Ca 2+ in the activity of Mycobacterium tuberculosis DNA gyrase

Karkare

Yousafzai

Mitchenall

et al. 2012

Nucleic Acids Research

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“…(21) This caveat is one of the major hurdles in studying calcium-signaling networks. In the case of the bacterial two-hybrid assay intracellular free calcium in E. coli is tightly regulated between 100 and 300 nM, 22,23 well below the reported calcium dissociation constants for S100B ( K d ∼ 20–60 μM) and most other S100 proteins. 2,3,24 This suggests the interaction of S100B with the D2 receptor interaction could be calcium independent.…”

mentioning

confidence: 78%

“…(21) The recent identification of the dopamine D2 receptor as an S100B target protein using a bacterial two-hybrid assay(20) presents one of the first cases where a binding partner for S100B has been identified using these methods. Since intracellular free calcium is tightly regulated near 100–300 nM in E. coli (22,23) and 100–200 nM in humans,(25) well below the calcium dissociation constants for most S100 proteins, this suggested that S100B might be interacting with the dopamine D2 receptor in the calcium-free state.…”

Section: Discussionmentioning

confidence: 99%

Identification of Calcium-Independent and Calcium-Enhanced Binding between S100B and the Dopamine D2 Receptor

Dempsey

Shaw

2011

Biochemistry

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S100B is a dimeric EF-hand protein that undergoes a calcium-induced conformational change and exposes a hydrophobic protein-binding surface. Recently S100B was identified as a binding partner of the dopamine D2 receptor in a bacterial two-hybrid screen involving the third intracellular loop (IC3). The low in vivo calcium concentration in bacteria (100–300 nM) suggests this interaction may occur in the absence of calcium. In this work the calcium-sensitive ability for S100B to recruit the IC3 of the dopamine D2 receptor was examined, and regions in both proteins required for complex formation were identified. Peptide array experiments identified the C-terminal 58 residues of the IC3 (IC3-C58) as the major interacting site for S100B. These experiments along with pull-down assays showed the IC3 interacts with S100B in the absence and presence of calcium. 1H–15N HSQC experiments were used to identify residues, primarily in helices III and IV, utilized in the IC3-C58 interaction. NMR titration data indicated that although an interaction between apo-S100B and IC3-C58 occurs without calcium, the binding was enhanced more than 100-fold upon calcium binding. Further, it was established that shorter regions within IC3-C58 comprising its N- and C-terminal halves had diminished binding to Ca2+-S100B and did not display any observable affinity in the absence of calcium. This indicates that residue or structural components within both regions are required for optimal interaction with Ca2+-S100B. This work represents the first example of an S100B target that interacts with both the apo- and calcium-saturated forms of S100B.

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“…Most studies estimate the intracellular calcium concentration in prokaryotes to be from 200 to 300 nM (46, 47), which is much lower than both the millimolar concentrations of Ca 2+ required to activate RpnA in vitro (Fig. S8E) and the concentration of cytosolic Mg 2+ (33).…”

Section: Discussionmentioning

confidence: 99%

Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

2017

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Bacteria use a variety of DNA-mobilizing enzymes to facilitate environmental niche adaptation via horizontal gene transfer. This has led to real-world problems, like the spread of antibiotic resistance, yet many mobilization proteins remain undefined. In the study described here, we investigated the uncharacterized family of YhgA-like transposase_31 (Pfam PF04754) proteins. Our primary focus was the genetic and biochemical properties of the five Escherichia coli K-12 members of this family, which we designate RpnA to RpnE, where Rpn represents recombination-promoting nuclease. We employed a conjugal system developed by our lab that demanded RecA-independent recombination following transfer of chromosomal DNA. Overexpression of RpnA (YhgA), RpnB (YfcI), RpnC (YadD), and RpnD (YjiP) increased RecA-independent recombination, reduced cell viability, and induced the expression of reporter of DNA damage. For the exemplar of the family, RpnA, mutational changes in proposed catalytic residues reduced or abolished all three phenotypes in concert. In vitro, RpnA displayed magnesium-dependent, calcium-stimulated DNA endonuclease activity with little, if any, sequence specificity and a preference for double-strand cleavage. We propose that Rpn/YhgA-like family nucleases can participate in gene acquisition processes.IMPORTANCE Bacteria adapt to new environments by obtaining new genes from other bacteria. Here, we characterize a set of genes that can promote the acquisition process by a novel mechanism. Genome comparisons had suggested the horizontal spread of the genes for the YhgA-like family of proteins through bacteria. Although annotated as transposase_31, no member of the family has previously been characterized experimentally. We show that four Escherichia coli K-12 paralogs contribute to a novel RecA-independent recombination mechanism in vivo. For RpnA, we demonstrate in vitro action as a magnesium-dependent, calcium-stimulated nonspecific DNA endonuclease. The cleavage products are capable of providing priming sites for DNA polymerase, which can enable DNA joining by primer-template switching.

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An assessment of the role of intracellular free Ca2+ in E. coli

Cited by 88 publications

References 41 publications

The role of Ca 2+ in the activity of Mycobacterium tuberculosis DNA gyrase

The role of Ca 2+ in the activity of Mycobacterium tuberculosis DNA gyrase

Identification of Calcium-Independent and Calcium-Enhanced Binding between S100B and the Dopamine D2 Receptor

Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

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