Biology subjects

Oeztuerk, M. A.

Publications and source records attributed to Oeztuerk, M. A..

3 recordsLinked to original sources

bioRxiv2020

DNA-sequence dependent positioning of the linker histone in a chromatosome: a single-pair FRET study

Linker histones (LH) bind to nucleosomes with their globular domain (gH) positioned in either an on- or an off-dyad binding mode. Here, we study the effect of the linker DNA (L-DNA) sequence on the binding of a full-length LH, Xenopus laevis H1.0b, to a Widom 601 nucleosome core particle (NCP) flanked by two 40 bp long L-DNA arms, by single-pair FRET spectroscopy. We varied the sequence of the 11 bp of L-DNA adjoining the NCP on either side, making the sequence either A-tract, purely GC, or mixed, with 64% AT. The labelled gH consistently exhibited higher FRET efficiency with the labelled L-DNA containing the A-tract, than that with the pure-GC stretch, even when the stretches were swapped. However, it did not exhibit higher FRET efficiency with the L-DNA containing 64% AT-rich mixed DNA when compared to the pure-GC stretch. We explain our observations with a model that shows that the gH binds on-dyad and that two arginines mediate recognition of the A-tract via its characteristically narrow minor groove. To investigate whether this on-dyad minor groove-based recognition was distinct from previously identified off-dyad major groove-based recognition, a nucleosome was designed with A-tracts on both the L-DNA arms. One A-tract was complementary to thymine and the other to deoxyuridine. The major groove of the thymine-tract was lined with methyl groups that were absent from the major groove of the deoxyuridine tract. The gH exhibited similar FRET for both these A-tracts, suggesting that it does not interact with the thymine methyl groups exposed on the major groove. Our observations thus complement previous studies that suggest that different LH isoforms may employ different ways of recognizingff AT-rich DNA and A-tracts. This adaptability may enable the LH to universally compact scaffold-associated regions and constitutive heterochromatin, which are rich in such sequences. Statement of SignificanceLinker histones (LHs) associate with the smallest repeat unit of chromatin, the nucleosome. They have been observed to have affinity for AT-rich DNA, which is found in constitutive heterochromatin and scaffold-associated regions (SAR), which could explain how the LHs can compact such parts of the chromatin. How the LH recognizes such sequences is poorly understood. Using single-pair FRET and modelling, we provide experimental evidence of DNA-sequence-induced changes in the orientation of a LH bound to a nucleosome, and thereby reveal a new mechanism by which the LH can recognize A-tract sequences that are abundantly present in the SAR. Our results show that, depending on how the LH associates with the nucleosome, it can employ more than one mechanism to recognize AT-rich DNA.

biophysics↗

bioRxiv2020

The active repertoire of Escherichia coli peptidoglycan amidases varies with physiochemical environment

Nearly all bacteria are encased in a peptidoglycan cell wall, an essential crosslinked matrix of polysaccharide strands and short peptide stems. In the Gram-negative model organism Escherichia coli, more than forty cell wall synthases and autolysins coordinate the growth and division of the peptidoglycan sacculus in the periplasm. The precise contribution of many of these enzymes to cell wall metabolism remains unclear due to significant apparent redundancy, particularly among the cell wall autolysins. E. coli produces three major LytC-type-N-acetylmuramoyl-L-alanine amidases, which share a role in separating the newly formed daughter cells during cytokinesis. Here, we reveal two of the three amidases exhibit growth medium-dependent changes in activity. Specifically, we report acidic growth conditions stimulate AmiB--and to a lesser extent, AmiC--activity. Combining computational and genetic analysis, we demonstrate that low pH-dependent stimulation of AmiB requires three periplasmic amidase activators: EnvC, NlpD, and YgeR. Altogether, our findings support overlapping, but not redundant, roles for the E. coli amidases in cell separation and illuminate the physiochemical environment as an important mediator of cell wall enzyme activity. IMPORTANCEPenicillin and related {beta}-lactam antibiotics targeting the bacterial cell wall synthesis are among the most commonly prescribed antimicrobials worldwide. However, rising rates of antibiotic resistance and tolerance jeopardize their continued clinical use. Development of new cell wall active therapeutics, including those targeting cell wall autolysins, has been stymied in part due to high levels of apparent enzymatic redundancy. In this study, we report a subset of E. coli amidases involved in cell separation during cell division are not redundant and instead are preferentially active during growth in distinct pH environments. Specifically, we discover E. coli amidases AmiB and AmiC are activated by acidic pH. Three semi-redundant periplasmic regulators--NlpD, EnvC, and YgeR--collectively mediate low pH-dependent stimulation of amidase activity. This discovery contributes to our understanding of how the cell wall remains robust across diverse environmental conditions and reveals opportunities for the development of condition-specific antimicrobial agents.

microbiology↗

bioRxiv2020

An inducible AraC that responds to blue light instead of arabinose

In Escherichia coli, the operon responsible for the catabolism of L-arabinose is regulated by the dimeric DNA-binding protein AraC. In the absence of L-arabinose, AraC binds to the distal I1 and O2 half-sites, leading to repression of the downstream PBAD promoter. In the presence of the sugar, the dimer changes conformation and binds to the adjacent I1 and I2 half-sites, resulting in the activation of PBAD. Here we engineer blue light-inducible AraC dimers in Escherichia coli (BLADE) by swapping the dimerization domain of AraC with blue light-inducible dimerization domains. Using BLADE to overexpress proteins important for cell shape and division site selection, we reversibly control cell morphology with light. We demonstrate the exquisite light responsiveness of BLADE by employing it to create bacteriographs with an unprecedented quality. We then employ it to perform a medium-throughput characterization of 39 E. coli genes with poorly defined or completely unknown function. Finally, we expand the initial library and create a whole family of BLADE transcription factors (TFs), which we characterize using a novel 96-well light induction setup. Since the PBAD promoter is commonly used by microbiologists, we envisage that the BLADE TFs will bring the many advantages of optogenetic gene expression to the field of microbiology.

microbiology↗